High efficiency and selectivity of recombinant proteins

Abstract

The present invention provides a novel expression system comprising a mammalian selectable marker that promotes the desired post-translational modification of a glycoprotein. Specifically, the present invention includes methods and compositions for achieving optimal recombinant protein expression in mammalian cells by using a selectable marker system based on a GPT gene of mammalian origin. The present invention includes methods for promoting the selectivity of recombinant proteins and increasing expression copies and protein yields in mammalian cells, and methods for using the GPT expression system.

Description

[0001] This application is a divisional application. The application number of the parent invention patent application is 201580044100.7, the application date is August 19, 2015, the priority date is August 19, 2014, and the name of the invention is “High-efficiency selectivity of recombinant proteins”. Background Art

[0002] Sequence Listing

[0003] This application incorporates by reference a Sequence Listing filed in computer readable form, created on August 3, 2015 as file 8700WO_ST25.txt (75,769 bytes). Technical Field

[0004] The present invention provides for the expression of recombinant proteins in mammalian cells in a consistent and efficient manner. Specifically, the present invention includes methods and compositions for achieving improved protein expression in mammalian cells by employing mammalian selection markers. The present invention includes methods for promoting the selectivity of recombinant proteins and increasing expression copy number and protein yield in mammalian cells, and methods for using such expression systems.

[0005] Description of related fields

[0006] The development of cell expression systems is an important goal to provide a reliable and efficient source of a given protein for research and therapeutic use. Recombinant protein expression in mammalian cells is often preferred for the preparation of therapeutic proteins due to, for example, the ability of mammalian expression systems to perform appropriate post-translational modifications on recombinant proteins.

[0007] There are a variety of vectors available for expression in mammalian hosts, each of which contains a selectable marker that allows easy isolation of cells expressing the recombinant protein during cell culture. Selectable marker genes (SMGs) are utilized in such systems because they confer a selective advantage to cells expressing the protein of interest, however, SMGs must be optimized for reasons such as phenotypic neutrality, high efficiency, and pluripotency.

[0008] Although many vectors and expression systems for hosting SMGs are available, the expression of recombinant proteins achieved in mammalian systems is generally unsatisfactory in terms of quantity, quality, or both. The biological "fingerprint" of a molecule (e.g., post-translational modifications, such as glycosylation) is particularly important in defining the utility and efficacy of a molecule in the development of recombinant protein therapeutics (Cumming, DA, 1990, Glycobiology, 1(2): 115-130). SMGs that do not negatively affect the biological properties of the expressed protein of interest are particularly advantageous.

[0009] Most SMGs are of bacterial origin and present additional disadvantages for use in mammalian systems due to growing concerns about the risk of lateral transfer of bacterial antibiotic resistance genes to environmental bacteria (Breyer, D. et al., 2014, Critical Reviews in Plant Sciences 33:286-330). Eliminating the use of bacterial antibiotic resistance genes may have a positive effect on consumer acceptance and mitigate such perceived risks.

[0010] Genetically engineered autologous cells have achieved rapid clinical success (see, e.g., Kershaw, MH et al., 2013, Nature Reviews: Cancer 13:525-541). The selection and design of vectors for genetic modification in human autologous cell products is critical, particularly because the undesired introduction of non-human components into human autologous cells may have serious consequences for patient safety (Eaker et al. 2013, Stem cells Trans. Med. 2:871–883; first published online in SCTM EXPRESS on October 7, 2013). A vector system with only mammalian-derived, rather than bacterial-derived, components would be advantageous for patient-specific T cells for adoptive immunotherapy.

[0011] Therefore, it is desirable to introduce mammalian selective genes, particularly those genes that provide phenotypic or metabolic advantages to transformed cells in expression systems for producing mammalian proteins of interest. In addition, cell lines that reliably express sufficiently high levels of therapeutic proteins and that carry out appropriate and consistent post-translational modifications to the therapeutic proteins are highly desirable. Therefore, there is a need in the art for improved mammalian expression systems. Summary of the invention

[0012] The use of mammalian tunicamycin (Tn) resistance genes as selective markers in mammalian expression systems can increase the efficiency and copy number of transfectants. It has been observed that the use of Tn resistance genes operably linked to the gene of interest creates selective pressure on mammalian cell populations, thereby increasing the random integration of transfectants (i.e., the gene of interest). It should be understood that the selective marker system can promote the selection of desired transfectants, but the method of the present invention gives an unexpected increase in the efficiency and random integration of the gene of interest, as well as a reliable biological quality of the desired protein. Therefore, the compositions and methods of the present invention make it possible to achieve a favorable selection of post-translational modifications that are good in quality of the expressed protein.

[0013] In one aspect, the invention provides an isolated cell comprising a mammalian tunicamycin (Tn) resistance gene, which encodes a protein having at least 93% identity to the amino acid sequence of SEQ ID NO: 3, which is operably linked to a gene of interest (GOI) and at least one regulatory element.

[0014] In another aspect, the present invention provides a method for preparing a recombinant protein of interest (POI), wherein the method comprises: providing a mammalian host cell encoding a nucleic acid molecule comprising (i) a mammalian tunicamycin (Tn) resistance gene and (ii) a gene encoding the POI; culturing the cell in the presence of a first concentration of Tn; isolating a population of cells expressing at least one copy of the Tn resistance gene; culturing the population of cells in the presence of increasing concentrations of Tn, wherein increasing the concentration of Tn increases production of the POI; and isolating the POI from the cell culture.

[0015] In yet another aspect, the invention provides a method for glycosylation of an N-glycan protein substrate, wherein the method comprises: providing a mammalian host cell encoding a nucleic acid molecule comprising a mammalian tunicamycin (Tn) resistance gene operably linked to a gene encoding a protein substrate to be glycosylated; culturing the cell in the presence of a first concentration of Tn; isolating a population of cells expressing at least one copy of the Tn resistance gene; culturing the population of cells in the presence of increasing concentrations of Tn, wherein increasing concentrations of Tn increase production of the POI; and isolating the protein substrate from the cell culture.

[0016] In some embodiments of the methods, the Tn resistance gene is operably linked to a gene encoding a POI, and the gene encoding a POI is operably linked to at least one regulatory element.

[0017] In some embodiments, the Tn resistance gene is exogenously added to the cell. In other embodiments, the Tn resistance gene encodes a protein having at least 93% identity to the amino acid sequence of SEQ ID NO: 3. In other embodiments, the Tn resistance gene encodes a protein having at least 94% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the Tn resistance gene encodes a protein having at least 93% identity to the amino acid sequence of SEQ ID NO: 4. In yet other embodiments, the Tn resistance gene encodes a protein having at least 94% identity to the amino acid sequence of SEQ ID NO: 4.

[0018] In some embodiments, the mammalian Tn resistance gene comprises a Chinese hamster (Cricetulus griseus) Tn resistance gene. In other embodiments, the mammalian Tn resistance gene comprises a human Tn resistance gene.

[0019] The Tn resistance gene may also include a nucleic acid sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17.

[0020] In certain embodiments of the aforementioned invention, the mammalian Tn resistance gene comprises a nucleic acid sequence that is at least 92% identical to the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the mammalian Tn resistance gene comprises a nucleic acid sequence that is at least 92% identical to the nucleic acid sequence of SEQ ID NO:12.

[0021] In the isolated cell of the present invention, at least one regulatory element operably linked to a Tn resistance gene is provided, wherein the regulatory element includes, but is not limited to, a promoter, a ribosome binding site, and an enhancer. In yet another embodiment, the GOI is operably linked to a promoter. In another embodiment, the GOI is operably linked to a ribosome binding site, such as an IRES.

[0022] In some embodiments, the isolated cells and methods of the present invention further comprise a second gene of interest (GOI), and the GOI encodes a protein of interest (POI). In one embodiment, the gene of interest (GOI) is an exogenously added GOI. In another embodiment, the exogenously added GOI is a human gene. In yet another embodiment, the regulatory element is an exogenously added regulatory element.

[0023] In other embodiments, the first and / or second GOI encodes a POI including but not limited to an antibody heavy chain, an antibody light chain, an antigen binding fragment and / or an Fc fusion protein.

[0024] In another embodiment, the first GOI and the second GOI are independently selected from genes encoding antibody light chains or antigen-specific fragments thereof, antibody heavy chains or antigen-specific fragments thereof, Fc fusion proteins or fragments thereof, and receptors or ligand-specific fragments thereof. In one embodiment, the recombinase recognition site is present between the first GOI and the second GOI. In other embodiments, the present invention also provides a recombinase recognition site at 5' of the first GOI and a recombinase recognition site at 3' relative to the second GOI.

[0025] In yet another embodiment, the GOI encodes a glycoprotein selected from an antibody light chain or an antigen binding fragment thereof, an antibody heavy chain or an antigen binding fragment thereof, an Fc fusion protein or a fragment thereof, a ligand, and a receptor or a ligand binding fragment thereof.

[0026] The isolated, non-naturally occurring cells of the present invention can be derived from eukaryotic cells. In one embodiment, the cell is a mammalian cell. In some embodiments, the isolated cell is an ex vivo human cell. In other embodiments, the cell is selected from: CHO (e.g., CHO K1, DXB-11CHO, Veggie-CHO), COS (e.g., COS-7), lymphocytes, stem cells, retinal cells, Vero, CV1, kidney (e.g., HEK293, 293EBNA, MSR 293, MDCK, HaK, BHK21), HeLa, HepG2, WI38, MRC 5, Colo25, HB 8065, HL-60, Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT cells, tumor cells, and cell lines derived from the aforementioned cells. In certain embodiments, the isolated cells of the invention are CHO-K1 cells, lymphocytes, retinal cells, or stem cells.

[0027] In one embodiment, the first concentration of Tn is 1 μg / mL. In another embodiment, the increasing concentrations of Tn include second and third concentrations of Tn.

[0028] In some embodiments, the second concentration is greater than the first concentration of Tn, and the third concentration is greater than the second concentration of Tn. In certain embodiments, the second concentration of Tn is 2.5 μg / ml, and the third concentration is 5 μg / mL.

[0029] In yet other embodiments, the increasing concentration of Tn comprises a second concentration of Tn, wherein the second concentration of Tn is 2.5 μg / ml or 5 μg / mL.

[0030] Unless otherwise stated or apparent from the context, any aspect or embodiment of the invention may be used in conjunction with any other aspect or embodiment of the invention.

[0031] Other objects and advantages will become apparent by reviewing the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram illustrating operable expression cassettes in a cloning vector structure for introducing a nucleic acid sequence encoding a gene of interest (e.g., eGFP) into the genome of a cell. SV40 promoter: simian virus 40 promoter; GPT: GlcNAc-1-P transferase (e.g., CHO-GPT, SEQ ID NO: 2; or hGPT, SEQ ID NO: 12); IRES: internal ribosome entry site; eGFP: enhanced green fluorescent protein; SV40 polyA: simian virus 40 polyA.

[0033] Figures 2A to 2C Represents mammalian GPT amino acid sequences, i.e., human (GPT_HUMAN; UniProtKB Accession No. Q9H3H5; SEQ ID NO:4), rhesus macaque (GPT_MACMU; UniProtKB Accession No. F6TXM3; SEQ ID NO:5), chimpanzee (GPT_PANTR; UniProtKB Accession No. H2R346; SEQ ID NO:6), dog (GPT_CANFA; UniProtKB Accession No. E2RQ47; SEQ ID NO:7), guinea pig (GPT_CAVPO; UniProtKB Accession No. E2RQ47; SEQ ID NO:8), rat (GPT_RAT; UniProtKB Accession No. Q6P4Z8; SEQ ID NO:9), and mouse (GPT_MOUSE; UniProtKB Accession No. P42867; SEQ ID NO:10). NO:10) compared with the Chinese hamster (GPT_CRIGR; UniProtKB accession number P24140; SEQ ID NO:3) GPT amino acid sequence.

[0034] Figure 3A and 3B Examples are given of how protein optimization can be achieved using the methods and compositions of the invention. Figure 3A Depicts a method for selecting positive cell transfectants from a first pool of cells cultured with 1 μg / mL tunicamycin (Tn). Subsequently, a second pool of cells is cultured with increasing concentrations of tunicamycin (eg, 2.5 μg / mL or 5 μg / mL) to enhance protein expression. Figure 3B Depicted is a method for selecting positive cell transfectants from a first pool of cells cultured with 1 μg / mL tunicamycin (Tn), and then serially increasing the Tn concentration in subsequent cell cultures in order to optimize protein expression.

[0035] Figures 4A to 4B The modified CHO cells include a YFP gene flanked by lox sites. Selectable markers (antibiotic resistance genes and eGFP) flanked by lox sites are incorporated into the YFP site and replaced with Cre recombinase via targeted integration. Random integrants express both YFP and eGFP. Figure 4A : Cells were transfected with Cre recombinase vector and hpt expression vector including eGFP; however, hygromycin was not present during culture. Figure 4B : Cells were transfected with Cre recombinase vector and hpt expression vector including eGFP; 400 μg / mL hygromycin was present.

[0036] Figures 5A to 5FFACS dot plots showing various parameters representing tunicamycin selectivity. Modified CHO cells include a YFP gene flanked by lox sites. Selectable markers (antibiotic resistance genes and eGFP) flanked by lox sites are incorporated at the YFP site and replaced with Cre recombinase via targeted integration. Random integrants express both YFP and eGFP. Figure 5A : Cells were transfected with Cre recombinase vector and CHO-GPT expression vector including eGFP; however, tunicamycin was not present during culture. Figure 5B : Cells were transfected with Cre recombinase vector and CHO-GPT expression vector including eGFP; 1 μg / mL tunicamycin was present. Figure 5C : Cells were transfected with Cre recombinase vector and CHO-GPT expression vector including eGFP; 2.5 μg / mL tunicamycin was present. Figure 5D : Cells were transfected with Cre recombinase vector and human GPT expression vector including eGFP; however, tunicamycin was not present during culture. Figure 5E : Cells were transfected with Cre recombinase vector and human GPT expression vector including eGFP; 1 μg / mL Tn was present. Fig. 5F : Cells were transfected with Cre recombinase vector and human GPT expression vector including eGFP; 2.5 μg / mL Tn was present.

[0037] Fig. 6A and 6B Shown is a comparison of a pool of GPT expressing cells to a non-GPT expressing pool in terms of their relative ability to enhance expression of an operably linked GOI, such as eGFP. Fig. 6A The relative gene copy numbers of CHO-GPT measured by PCR for the cell pools are illustrated as follows: Pool-49 cells without Tn selection (no exogenous GPT added); Pool-49 cells with 5 μg Tn selection (no exogenous GPT); Pool-1 cells naturally express higher amounts of GPT (data not shown) and were tested without Tn selection; Pool-78 cells without Tn selection (no exogenous GPT); CHO cells expressing exogenously added hpt and selected with 400 μg / mL hygromycin; CHO cells expressing exogenous GPT under 1 μg / mL Tn selection conditions; CHO cells expressing exogenous GPT selected from the 1 μg / mL Tn selection pool were further cultured in 1 μg / mL Tn; CHO cells expressing exogenous GPT selected from the 1 μg / mL Tn selection pool were further cultured in 2.5 μg / mL Tn; CHO cells expressing exogenous GPT selected from the 1 μg / mL Tn selection pool were further cultured in 2.5 μg / mL Tn; CHO cells expressing exogenous GPT selected by the Tn selection pool were further cultured in 5 μg / mL Tn. Figure 6B The same cell pool (such as Fig. 6A) The relative number of gene copies of the gene of interest eGPT measured.

[0038] Figures 7A to 7D The glycoform characteristics of Fc fusion protein 1 (FcFP1) produced by cell culture are described as follows: Fig. 7A : CHO cells not expressing GPT (batch B10002M410) using standard protocol, compared to Figure 7B : CHO cells expressing CHO-GPT without Tn selection (batch 110728). Figure 7C : CHO cells expressing CHO-GPT and selected with 1 μg / mL Tn (batch 110728-01), compared to Fig.7D : CHO cells expressing CHO-GPT and selected with 5 μg / mL Tn (batch 110728-02). Each chromatogram indicates the fraction containing sialylated residues as follows: OSA=0 sialic acid residues; 1SA=1 sialic acid residue; 2SA=2 sialic acid residues; 3SA=3 sialic acid residues; 4SA=4 sialic acid residues.

[0039] Figure 8 Illustrate the superimposed glycosylation profiles of Fc fusion protein 1 (FcFP1) sampled from (A) batch B10002M410, (B) batch 110728, (C) batch 110728-01, and (D) batch 110728-02. The glycosylation profiles of each protein produced from the GPT batches were compatible with the reference standard protein and consistently produced major glycoform species. It is apparent that no new and unique glycoform species were produced in the GPT batches compared to the reference standard protein. DETAILED DESCRIPTION

[0040] Before describing the method of the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can vary. It should also be understood that the terminology used herein is only used for the purpose of describing specific embodiments, and is not intended to be limiting, because the scope of the present invention will be limited only by the appended claims.

[0041] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one or more methods and / or one or more steps of the type described herein and / or which will become apparent to one skilled in the art upon reading this disclosure.

[0042] Unless defined otherwise or otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0043] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, specific methods and materials are now described.All publications mentioned herein are incorporated by reference in their entirety.

[0044] A variety of genes well known in the art can confer selective phenotypes to mammalian cells in culture. Generally speaking, the selective marker gene expresses a protein, usually an enzyme that confers resistance to various antibiotics in cell culture. Under certain selection conditions, cells expressing fluorescent protein markers can be visualized and are therefore selectable. Examples in the art include beta-lactamase (bla; beta-lactam antibiotic resistance gene or ampR; ampicillin resistance gene), bls (blasticidin resistance acetyltransferase gene), hygromycin phosphotransferase (hpt; hygromycin resistance gene), etc.

[0045] The methods described herein rely on the use of tunicamycin and an enzyme (marker) that allows cells to grow in cell culture by making them resistant to tunicamycin. Tunicamycin (Tn) is a mixture of antibiotics that acts as an inhibitor of bacterial and eukaryotic N-acetylglucosamine transferases, thereby preventing the formation of N-acetylglucosamine lipid intermediates and the glycosylation of newly synthesized glycoproteins. (King, IA and Tabiowo, A., 1981, Effect of tunicamycin on epidermal glycoprotein and glycosaminoglycan synthesis in vitro. Biochem. J., 198(2): 331-338). Tn is cytotoxic because it specifically inhibits UDP-N-acetylglucosamine:phospho-diprol N-acetylglucosamine-1-P transferase (GPT), an enzyme that catalyzes the initial step in the biosynthesis of dolichol-linked oligosaccharides. In the presence of tunicamycin, asparagine-linked glycoproteins produced in the endoplasmic reticulum (ER) are not glycosylated by N-linked glycans and therefore may not fold properly within the ER and thus may be degraded (Koizumi et al. 1999, Plant Physiol. 121(2):353–362). Thus, Tn is an inducer of the notable unfolded protein response (UPR) in bacteria and eukaryotic cells that leads to apoptosis.

[0046] The gene for uridine diphosphate GPT (also known as GlcNAc-1-P transferase) was identified as being overexpressed under certain cellular conditions to confer resistance to Tn (Criscuolo and Krag, 1982, J Biol Chem, 263(36):19796-19803; Koizumi et al., 1999, Plant Physiology, Vol. 121, pp. 353-361). The gene encoding GPT (also described as GenBank Accession No. M36899 (SEQ ID NO: 2)) was isolated from a Tn-resistant Chinese hamster ovary cell line and encodes a 408 amino acid protein (SEQ ID NO: 3) (Scocca and Krag, 1990, J Biol Chem 265(33):20621-20626; Lehrman, M. et al., 1988, J Biol Chem 263(36):19796-803). Hamster GPT is overexpressed in yeast cells (S. pombe) and confers Tn resistance in these cells; a convenient source of purified GPT enzyme is also provided (Scocca JR et al. 1995, Glycobiology, 5(1): 129-36). The transcription level of GPT was analyzed in hybridoma cells (IgG-expressing B cells, compared to quiescent B cells), and although IgG-producing cells were observed not to show elevated levels of GPT transcription or activity, a small increase in GPT was seen in B cells transitioning from quiescence to activation. It was concluded that GPT levels may correspond to the early generation of a proliferative response to LPS (antigen) stimulation in B cells (Crick, DC et al. 1994, J Biol Chem 269(14): 10559-65).

[0047] Furthermore, it was previously unclear whether altering the expression of GPT in the presence or absence of Tn in a cell expression system would have an impact on the glycosylation of the protein product and thus affect product quality. It is understood that optimal and consistent glycosylation is a critical protein attribute in the preparation of therapeutic glycoproteins.

[0048] The present invention provides an improved method for producing recombinant proteins in a mammalian cell system using the mammalian Tn resistance gene GPT as a regulatable selection marker, wherein increased copy number of a gene of interest operably linked to GPT is associated with increased random integration of the GPT expression cassette into the cell.

[0049] It is recognized in the art that the preparation of therapeutic proteins, especially glycoproteins, relies on mammalian expression systems that mimic the natural glycosylation of such proteins. (For a review, see Bork, K. et al., 2009, J Pharm Sci. 98 (10): 3499-3508). For example, the terminal monosaccharides of certain glycoproteins (such as N-linked complex glycans) are usually occupied by sialic acid. Sialylation can affect the pharmacokinetic properties of glycoproteins, such as absorbance, serum half-life and clearance or other physicochemical or immunogenic properties of glycoproteins. Overexpressed recombinant glycoproteins often have incomplete or inconsistent glycosylation. Reliable methods are essential for process consistency and quality of therapeutic glycoproteins produced in mammalian cell lines.

[0050] The present invention also provides improved methods for glycosylation of recombinant proteins, ie, methods for producing glycoproteins in mammalian cell systems, so as to provide consistent mass yields of the desired protein.

[0051] definition

[0052] DNA regions are operably linked when they are functionally related to each other. For example, a promoter is operably linked to a coding sequence if it is able to participate in the transcription of the sequence; a ribosome binding site is operably linked to a coding sequence if it is positioned so that it can be translated. In general, operably linked includes but is not necessarily adjacent. For sequences such as secretory leader sequences, proximity and proper placement in the reading frame are typical features. Enhancement-generated sequences (such as promoters) are operably linked to a gene of interest (GOI) when they are functionally related to the GOI, for example, when their presence causes increased expression of the GOI.

[0053] Thus, the phrase "operably linked" (such as in the context of a DNA expression vector construct) control sequences (e.g., promoters or operators or markers) are appropriately located at a position relative to the coding sequence such that the control sequence directs or permits the production of the polypeptide / protein of interest encoded by the coding sequence. For example, when a selectable marker is necessary for cell survival under certain culture conditions, the gene of interest is operably linked to the selectable marker gene because expression will not occur without the presence of an operable selectable marker protein.

[0054] As used herein, "promoter" refers to a DNA sequence sufficient to direct the transcription of a DNA sequence to which it is operably linked, i.e., its connection mode allows transcription of a gene of interest and / or a selectable marker gene when appropriate signals are present. The expression of a gene may be placed under the control of any promoter or enhancer element known in the art.

[0055] "Expression vector" in the context of the present invention can be any suitable vector, including chromosome, non-chromosome and synthetic nucleic acid vector (including a set of nucleic acid sequences of suitable expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from plasmid and phage DNA combinations, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the nucleic acid molecule encoding Fc fusion protein or polypeptide is included in a naked DNA or RNA vector (including, for example, linear expression elements (as described in, for example, Sykes and Johnston, 1997, Nat Biotech 12, 355-59)), a compact nucleic acid vector (as described in, for example, US6,077,835 and / or WO00 / 70087) or a plasmid vector such as pBR322, pUC 19 / 18 or pUC 118 / 119. Such nucleic acid vectors and their use are well known in the art (see, for example, US Pat. Nos. 5,589,466 and 5,973,972).

[0056] As used herein, "operator" refers to a DNA sequence that is introduced into or near a gene in such a way that the gene can be regulated by binding a repressor protein to the operator and thereby preventing or allowing transcription of the GOI (i.e., nucleotides encoding a polypeptide or protein of interest).

[0057] The ribosome binding site includes an "internal ribosome entry site" (IRES) or may include a 5' cap. Many IRES sequences are well known in the art. IRES stands for translation control sequence, wherein the IRES site is usually located 5' of the gene of interest and allows RNA to be translated in a cap-independent manner. The transcribed IRES can directly bind to the ribosomal subunit so that the position of the mRNA start codon is properly oriented in the ribosome for translation. The IRES sequence is usually located in the 5'UTR of the mRNA (just upstream of the start codon). IRES functionally replaces the need for various protein factors that interact with the eukaryotic translation machinery.

[0058] The term "enhanced" or "improved" when used to describe protein expression includes an increase in the amount and / or quality consistency of the protein (i.e., gene product) produced by the expression system or method of the present invention. Therefore, this includes an increase of at least about 1.5 times to an increase of at least about 3 times in expression compared to the results usually observed by random integration into the genome, for example, compared to the integrant pool using another selective marker construct. Therefore, the expression increase fold observed by the protein of interest is compared with the expression level of the same gene measured under substantially the same conditions in the absence of the expression cassette or cell of the present invention including the GPT gene, or in the presence of the expression cassette or cell including different selective markers. The expression increase can also be measured by the number of random integration events generated. The increased recombination efficiency includes the enhancement of the recombination ability of the locus (e.g., using a recombinase recognition site). Enhancement refers to the measurable efficiency compared to random recombination, which is generally 0.1%. Under certain conditions, the enhanced recombination efficiency is about 10 times, or about 1%, of random. Unless otherwise specified, the invention protected by the claims is not limited to a specific recombination efficiency. Enhanced expression can also be measured by the number of gene copies produced, such as by quantitative polymerase chain reaction (qPCR) or other well-known techniques.

[0059] Enhanced or improved products also refer to more consistent quality, such as post-translational modifications observed by the GPT expression system of the present invention. Consistent quality includes, for example, having a desired glycosylation profile after repeated production lines. Consistency refers to uniformity and standardization in terms of quality, while repeated production batches are essentially unchanged. Calculation of the Z number to measure consistency is taught herein. Other statistical indicators for measuring consistency are known in the art.

[0060] The phrase "selective pressure" is a force or stimulus applied to a living organism (eg, a cell) or a system (eg, an expression system) that alters the behavior and survival (eg, ability to survive) of the living organism or system in a given environment.

[0061] The phrase "gene amplification" refers to the increase in the number of identical copies of a gene sequence. Certain cellular processes are characterized by the production of multiple copies of one or more specific genes, which amplifies the phenotype conferred by the gene to the cell, such as antibiotic resistance.

[0062] When the phrase "exogenously added gene" or "exogenously added GOI" is used in reference to an expression cassette, the phrase refers to any gene not found in the genome of a cell in nature, or an additional copy of a gene integrated into the genome (in a different locus in the genome). For example, an "exogenously added gene" (e.g., a selectable marker gene) within the CHO genome may be a hamster gene not found in nature within a particular CHO locus (i.e., a hamster gene from another locus in the hamster genome), a gene from any other species (e.g., a human gene), a chimeric gene (e.g., human / mouse), or may be a hamster gene not found in nature within the CHO genome (i.e., a hamster gene that has less than 99.9% identity to a gene from another locus in the hamster genome), or any other gene not found in nature within the native genome of the CHO.

[0063] Random integration events are different from targeted integration events, and the genome of the gene inserted into the cell is not site-specific in random integration events. The example of targeted integration is homologous recombination. Random (non-homologous) integration represents that the position (locus) of the integrant produced is not known or specified. Random integration is considered to occur by non-homologous end joining (NHEJ), but is not limited to this method.

[0064] The selection efficiency expresses the percentage of the population of surviving cells that express the selectable marker and, where appropriate, the protein of interest under the control of the selectable marker.

[0065] Percent identity when describing a Tn resistance protein is intended to include homologous sequences that exhibit such identity along contiguous regions of homology, but the presence of gaps, deletions or insertions that do not have homology in the compared sequences is not included in the calculation of percent identity. In interpreting the use of "percent identity" in this context, the following amino acid sequence comparison will be referred to:

[0066]

[0067] As used herein, the determination of "percent identity" between the above "GPT_CRIG" sequence (for Chinese hamster GPT) and the mouse homolog ("GPT_mouse") should not include comparison of hamster amino acids 10 and 11 because the hamster homolog does not have a homologous sequence to compare in the alignment (i.e., the mouse GPT has an insertion at that point, or the hamster homolog has a gap or deletion, as the case may be). Therefore, in the above comparison, the percent identity comparison will extend from "MWA" at the 5' end to "ESQ" at the 3' end. In this event, the mouse homolog differs only in that it has an "R" at hamster GPT position 51. Because the comparison is made over a stretch of 58 contiguous bases in 60 base pairs, with only one amino acid difference (which is not a gap, deletion, or insertion), there is over 98% identity between the two sequences (mouse and hamster) from hamster GPT position 1 to hamster GPT position 58 (because "percent identity" does not include penalties for gaps, deletions, and insertions). Although the above examples are based on amino acid sequences, it will be appreciated that nucleic acid sequence percent identity will be calculated in the same manner.

[0068] The term "cell" includes any cell suitable for expressing a recombinant nucleic acid sequence. Cells include cells of prokaryotes and eukaryotes (single or multicellular), bacterial cells (e.g., strains of Escherichia coli (E. coli), Bacillus spp., Streptomyces spp., etc.), mycobacterial cells, fungal cells, yeast cells (e.g., Saccharomyces cerevisiae, S. pombe, Pichia pastoris, Pichia methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni, etc.), non-human animal cells, mammalian cells, human cells, or cell fusions such as hybridomas or quadromas. In certain embodiments, the cell is a human, monkey, ape, hamster, rat, or mouse cell. In other embodiments, the cell is a eukaryotic cell and is selected from the following cells: CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293EBNA, MSR 293, MDCK, HaK, BHK21), HeLa, HepG2, WI38, MRC 5, Colo25, HB 8065, HL-60, Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT cells, tumor cells, and cell lines derived from the foregoing cells. In some embodiments, the cell comprises one or more viral genes, such as retinal cells expressing viral genes (e.g., PER.C6 cell).

[0069] The phrase "cumulative cell density" or "ICD" refers to the cell density in a culture medium considered as a whole over a period of time, expressed as cell-days per milliliter. In some embodiments, the ICD is measured in cells at about day 12 in culture.

[0070] "Glycosylation" or the phrase "glycosylate a protein" includes the formation of glycoproteins with oligosaccharides attached to the side chains of asparagine (Asn) residues (i.e., N-linked) or serine (Ser) / threonine (Thr) residues (i.e., O-linked) of the protein. Glycans can be homopolymers or heteropolymers of monosaccharide residues, which can be linear or branched. N-linked glycosylation is known to initiate primarily in the endoplasmic reticulum, while O-linked glycosylation has been shown to initiate in the ER or Golgi apparatus.

[0071] "N-glycan proteins" or "N-glycan protein substrates" include proteins that contain or can accept N-linked oligosaccharides. N-glycans can be composed of N-acetylgalactosamine (GalNAc), mannose (Man), fructose (Fuc), galactose (Gal), neuraminic acid (NANA), and other monosaccharides, however, N-glycans generally have a common core pentasaccharide structure that includes: three mannose and two N-acetylglucosamine (GlcNAc) sugars. Proteins with the contiguous amino acid sequence (i.e., sequence stretch) Asn-X-Ser or Asn-X-Thr, where X is any amino acid except proline, can provide attachment sites for N-glycans.

[0072] General Description

[0073] The present invention is based, at least in part, on the discovery that under certain conditions a recombinant protein can be produced in cells wherein a gene encoding the protein is operably linked to a Tn resistance gene, GPT, and selection of cells producing the protein is configured to increase random integration events in the cell genome and thereby increase the copy number of the gene of interest and ultimately increase protein production.

[0074] The present invention is also based at least in part on the discovery that protein-producing cells can be optimized to express proteins with consistent and reliable post-translational modifications. The GPT expression cassette can also be integrated into the cell genome as in an expression construct, such as via an expression vector using various gene editing techniques known in the art. An expression vector comprising a GPT can be integrated into the genome by random or targeted recombination, such as homologous recombination or recombination mediated by a recombinase that recognizes a specific recombination site (e.g., Cre-lox mediated recombination).

[0075] Homologous recombination in eukaryotic cells can be promoted by introducing a break in chromosomal DNA at the integration site. Model systems have shown that if double-strand breaks are introduced in the chromosomal target sequence, the frequency of homologous recombination during gene targeting will increase. This can be achieved by targeting certain nucleases to specific integration sites. DNA binding proteins that recognize DNA sequences at the target locus are known in the art. Gene targeting vectors are also used to promote homologous recombination. In the absence of a gene targeting vector for homology directed repair (homology directed repair), cells often close the double-strand breaks by non-homologous end joining (NHEJ) (which may cause the deletion or insertion of multiple nucleotides at the cleavage site). Gene targeting vector construction and nuclease selection are within the technical scope of the technical personnel of the field to which the present invention belongs.

[0076] In some examples, zinc finger nucleases (ZFNs) with modular structures and containing individual zinc finger domains recognize specific 3-nucleotide sequences in a targeted sequence (e.g., a site of targeted integration). Some embodiments may utilize ZFNs with combinations of individual zinc finger domains targeting multiple target sequences.

[0077] Transcription activator-like (TAL) effector nucleases (TALENs) can also be used for site-specific genome editing. TAL effector protein DNA binding domains are often used in combination with non-specific cleavage domains of restriction nucleases such as FokI. In some embodiments, a fusion protein comprising a TAL effector protein DNA binding domain and a restriction nuclease cleavage domain is used to recognize and cleave DNA at a target sequence within the locus of the present invention (Boch J et al., 2009 Science 326: 1509-1512).

[0078] RNA-guided endonucleases (RGENs) are programmable genome engineering tools developed from bacterial adaptive immune mechanisms. In this system (clustered regularly spaced short palindromic repeats (CRISPR) / CRISPR correlation (Cas) immune response), protein Cas9 forms sequence-specific endonucleases when compounded with two RNAs (one of which guides target selection). RGENs are composed of components (Cas9 and tracrRNA) and target-specific CRISPR RNA (crRNA). The efficiency of DNA target cleavage and the position of the cleavage site are all based on the position of the protospacer sequence adjacent to the motif (PAM) and change, which is an additional requirement for target recognition (Chen, H. et al., J.Biol.Chem. March 14, 2014 as manuscript M113.539726 published online).

[0079] Other homologous recombination methods are available to technicians, such as BuD-derived nucleases (BuDN) with precise DNA binding specificity (Stella, S. et al. Acta Cryst. 2014, D70, 2042-2052). The precise genome modification method is selected based on the tools available for compatibility with the unique target sequence within the genome to avoid cell phenotype being destroyed.

[0080] The present invention provides cells and methods for stably integrating nucleic acid sequences (genes of interest) into mammalian cells, wherein the nucleic acid sequences can enhance expression by integration with GPT sequences. The present invention also provides compositions and methods for using GPT in connection with expression constructs (e.g., expression vectors), and for adding exogenous GPT to mammalian cells of interest. The present invention provides cells and methods for methods for consistent but robust production of glycoproteins, particularly therapeutic glycoproteins.

[0081] Construction of the GPT selection marker cassette

[0082] Provided herein are expression vectors comprising an operable GPT expression cassette. The expression cassette includes the necessary regulatory elements to allow and drive transcription and translation of mammalian GPT and the desired gene product.

[0083] Various combinations of genes and regulatory sequences described herein can also be developed. Examples of other combinations of suitable sequences described herein that can also be developed include sequences containing multiple copies of the GPT genes disclosed herein, or sequences derived by combining the disclosed GPT with other nucleotide sequences to achieve an optimal regulatory element combination. Such combinations can be connected or arranged in series to provide the optimal GPT spacing for orientation to the genes and regulatory elements of interest.

[0084] Homologous sequences of the gene encoding GPT are known to exist in cells from other mammalian species (such as humans; see Figure 2) and in cell lines derived from other mammalian tissue types, and can be isolated by techniques well known in the art. A list of exemplary mammalian GPT amino acid sequences is provided in Figure 2. The nucleotide sequences shown in SEQ ID NOs: 2 and 11-17 can be subjected to nucleotide sequence changes, such as codon optimization, to allow optimal expression of the corresponding GPT proteins shown in SEQ ID NOs: 3-10. In addition, the amino acid sequences shown in SEQ ID NOs: 3-10 can be altered by changing the nucleotide sequence encoding GPT. Such techniques include, but are not limited to, site-directed or random mutagenesis techniques well known in the art.

[0085] The resulting GPT variants can then be tested for GPT activity as described herein, such as resistance to tunicamycin. By routine experimentation, a GPT protein having an amino acid sequence at least about 93% identical, or at least about 95% identical, or at least about 96% identical, or at least about 97% identical, or at least about 98% identical to SEQ ID NO:3 having GPT activity is isolated and is expected to exhibit the same Tn resistance, selective efficiency, and post-translational benefits as SEQ ID NO:3. Thus, mammalian homologs of GPT and GPT variants are also encompassed by embodiments of the present invention. Figures 2A to 2C An alignment of various mammalian GPT amino acid sequences (ie, SEQ ID NOs: 3-10) is shown. Mammalian GPT sequences (nucleic acid and amino acids) are conserved among hamster, human, mouse, and rat genomes. Table 1 identifies exemplary mammalian GPT proteins and their degree of homology.

[0086] Table 1A: Amino Acid Identity of GPT Homologs

[0087]

[0088] Table 1B: Nucleic acid identities of representative GPT homologs

[0089] animal SEQ ID NO % Hamster Identity Hamster 2 - Mouse 11 92 Human 12 92 Rat 13 94 Macaque 14 92 chimpanzee 15 92

[0090] The GPT / tunicamycin method provided herein can be used to generate a cell population that expresses increased levels of a protein of interest. The absolute level of expression will vary with a particular protein, depending on how efficiently the protein is processed by the cell.

[0091] Therefore, the present invention also includes a nucleotide sequence expressing GPT selected from SEQ ID NO: 2 and 11-17. The present invention also encompasses a nucleotide sequence expressing GPT that is at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 98% identical, or at least 99% identical to a nucleotide sequence selected from SEQ ID NO: 2 and 11-17.

[0092] The present invention includes vectors comprising SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 12. Vectors comprising a mammalian GPT gene and optional regulatory elements include vectors for transient or stable transfection.

[0093] In one embodiment, the GPT gene is used to enhance the expression of the GOI, such as Figure 1 shown. Figure 1 A GOI is shown operably linked to an IRES sequence and a GPT selectable marker. The GPT cassette also includes a promoter sequence, such as the SV40 promoter, and a polyadenylation (poly(A)) sequence, such as the SV40 poly(A).

[0094] The expression enhancement cassette (including GPT and upstream promoter) is optimally integrated into the cell genome. Using the method of the present invention, GOI is expressed in the GPT expression cassette under culture conditions based on increasing concentrations of Tn ( Figure 3A or Figure 3B ). FACS readout results (such as Figure 5B , 5C , 5E and 5F) illustrate the expression profile in a stably transfected cell population, particularly the dramatic increase in selection efficiency using the mammalian Tn resistance selection markers CHO-GPT and hGPT. Mammalian GPT expression also enhances the expression of the gene product of interest, such as the production of the fluorescent protein eGFP. Compared to GOIs expressed in an expression system using GPT under culture conditions based on one concentration of Tn, continuous culture with increasing concentrations of Tn resulted in approximately 2-fold enhanced expression, such as Figure 6B Examples are given in .

[0095] The present invention includes mammalian cells comprising such a GPT gene, wherein the GPT gene is exogenous and integrated into the cell genome by the method of the present invention. Cells comprising such a GPT gene have at least one exogenously added gene of interest (GOI) that is upstream or downstream of the GPT gene.

[0096] In various embodiments, the expression of the GOI can be enhanced by placing the GOI under the control of a mammalian selective marker GPT. In other embodiments, the random integration event of the GOI can be enhanced by placing the GOI under the control of a mammalian selective marker GPT and providing cell culture conditions including a Tn concentration greater than 0.5 μg / mL. In some embodiments, the cell culture conditions include a Tn concentration greater than 1 μg / mL. The regulatory element can be operably linked to the GOI, wherein the expression of the GOI - at a selected distance between the GOI and the GPT (in the 5' or 3' direction) - retains the ability to enhance the expression of the GOI, for example, exceeding the expression usually observed due to random integration events. In various embodiments, the enhancement is at least about 1.5 times to about 2 times or more. Compared to random integration or random expression, the enhancement of expression is about 1.5 times or about 2 times or more.

[0097] In another embodiment, consistently glycosylated proteins can be obtained using the methods and compositions of the present invention. As shown in Table 4, the Tn-treated GPT / GOI recombinant protein batches resulted in identical glycosylation profiles for repeated batches. Thus, enhanced protein expression, such as consistent glycosylation profiles, can be directly compared by calculating the Z number as taught herein. The Z number equation takes into account the relative number of peaks on the chromatogram representing the sialic acid (SA) moiety and the relative shape and intensity of each peak. The Z number is based on the area occupied by each peak and can be used as an indicator of consistency of a complex glycoprotein (see, e.g., Figures 7A-7D , Figure 8 and Example 3, as described herein).

[0098] Protein expression optimization can also be achieved for each GOI, including, for example, expression cassette orientation or codon optimization. Protein optimization can also be achieved by varying the increasing Tn concentration in the cell culture method.

[0099] Recombinant expression vectors may include a synthetic or cDNA-derived DNA fragment encoding a protein, which may be operably linked to a suitable transcription and / or translation regulatory element derived from a mammal, virus or insect gene. Such regulatory elements include a transcription promoter, an enhancer, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling transcription and translation termination, as described in detail herein. Mammalian expression vectors may also include non-transcriptional elements, such as a replication origin, other 5' or 3' flanking non-transcribed sequences, and 5' or 3' non-translated sequences such as splicing donors and acceptor sites. Other selective marker genes (such as fluorescent markers) that help identify transfectants may also be incorporated.

[0100] In another embodiment, the vector includes a nucleic acid molecule encoding a protein of interest (or a gene of interest), which includes an expression vector containing the nucleic acid molecule (gene), wherein the nucleic acid molecule (gene) is operably linked to an expression control sequence.

[0101] Vectors are provided that include a gene of interest (GOI), wherein the GOI is operably linked to expression control sequences suitable for expression in a mammalian host cell.

[0102] Useful promoters for use in the present invention include, but are not limited to, the SV40 early promoter region, the promoter contained in the 3' long terminal repeat of Rous sarcoma virus, the regulatory sequences of the metallothionein gene, the mouse or human cytomegalovirus IE promoter (Gossen et al., (1995) Proc. Nat. Acad. Sci. USA 89:5547-5551), the cauliflower mosaic virus 35S RNA promoter, and the promoter of the photosynthetic enzyme ribulose bisphosphate carboxylase, promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, the PGK (phosphoglycerol kinase) promoter, the alkaline phosphatase promoter, and the following animal transcription control regions, which exhibit tissue specificity and have been used in transgenic animals: elastase I, insulin, immunoglobulins, mouse mammary tumor virus, albumin, alpha-fetoprotein, alpha 1-antitrypsin, beta-globulin, and myosin light chain-2.

[0103] The nucleic acid molecules of the present invention may also be operably linked to an effective poly (A) termination sequence (e.g., SV40 poly (A)), an origin of replication for plasmid products in E. coli, and / or a convenient cloning site (e.g., a polylinker). The nucleic acid may also include an adjustable inducible promoter (inducible, repressible, developmentally regulated) as opposed to a constitutive promoter, such as CMV IE (the skilled artisan will recognize that such terms are actually descriptors of gene expression levels under certain conditions).

[0104] The present invention provides methods for preparing proteins of interest, and provides expression vectors comprising genes of interest (GOI). Such expression vectors can be used for recombinant preparation of any proteins of interest. Transcription and translation control sequences in expression vectors that can be used to transfect vertebrate cells can be provided by viral sources. For example, commonly used promoters and enhancers are derived from viruses, such as polyoma virus, adenovirus 2, simian virus 40 (SV40) and human cytomegalovirus (CMV). Viral genomic promoters, control and / or signal sequences can be used to drive expression, and such control sequences provided are compatible with the selected host cells. Depending on the cell type in which the recombinant protein is expressed, non-viral cell promoters (e.g., β-globin and EF-1α promoters) can also be used.

[0105] DNA sequences derived from the SV40 viral genome, such as the SV40 origin, early and late promoters, enhancers, splicing and polyadenylation sites, can be used to provide other genetic elements useful for the expression of heterologous DNA sequences. Early and late promoters are particularly useful because both can be easily obtained from the SV40 virus as fragments that also contain the SV40 viral replication origin (Fiers et al., Nature 273:113, 1978). Smaller or larger SV40 fragments can also be used. Typically, about 250 bp of sequence extending from the Hind III site located in the SV40 replication origin to the BglI site is included.

[0106] Bicistronic expression vectors for expressing multiple transcripts have been previously described (Kim SK and Wold BJ, Cell 42:129, 1985; Kaufman et al. 1991, supra) and can be used in combination with the GPT expression system. Other types of expression vectors will also be useful, such as those described in U.S. Pat. No. 4,634,665 (Axel et al.) and U.S. Pat. No. 4,656,134 (Ringold et al.).

[0107] The integration site (e.g., recombinase recognition site) may be placed 5' or 3' to the gene sequence encoding the POI. An example of a suitable integration site is a lox p site. Another example of a suitable integration site is two recombinase recognition sites, for example selected from a lox p site, a lox and a lox 5511 site.

[0108] Gene amplification cassette and expression vector thereof

[0109] Useful regulatory elements previously described or known in the art may also be included in nucleic acids used to transfect mammalian cells. Figure 1 An operable cassette in a GPT vector is exemplified, which also includes a promoter sequence, an IRES sequence, a gene of interest, and a poly(A) sequence.

[0110] The expression vector in the context of the present invention can be any suitable vector, including chromosome, non-chromosome and synthetic nucleic acid vector (including a group of suitable expression control element nucleic acid sequence). The example of such vector includes derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from plasmids and phage DNA combinations, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the nucleic acid molecule encoding the antibody is contained in a naked DNA or RNA vector, including, for example, linear expression elements (such as described in, for example, Sykes and Johnston, Nat Biotech 12, 355-59 (1997)), compact nucleic acid vectors (such as described in, for example, US 6,077,835 and / or WO 00 / 70087) or plasmid vectors such as pBR322, pUC 19 / 18 or pUC 118 / 119. Such nucleic acid vectors and their use are well known in the art (see, eg, US 5,589,466 and US 5,973,972).

[0111] Alternatively, the expression vector may be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be used. Suitable vectors include, for example, vectors comprising constitutive or inducible promoters, such as yeast alpha factor, alcohol oxidase, and PGH (reviewed in: F. Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987) and Grant et al., Methods in Enzymol 153, 516-544 (1987)).

[0112] In certain embodiments, the vector includes a nucleic acid molecule encoding a protein of interest (or a gene of interest), which includes an expression vector containing the nucleic acid molecule (gene), wherein the nucleic acid molecule (gene) is operably linked to an expression control sequence suitable for expression in a host cell.

[0113] Expression control sequences are engineered to control and drive transcription of a gene of interest, and subsequent protein expression in various cell systems. Plasmids combine an expressible gene of interest with an expression control sequence (i.e., expression cassette) comprising desired regulatory elements such as promoters, enhancers, selectable markers, operators, etc. In the expression vectors of the present invention, GPTs and nucleic acid molecules encoding proteins of interest such as antibodies may include or be associated with any suitable promoter, enhancer, operator, repressor, poly(A) termination sequence, and other expression promoting elements.

[0114] The expression of the gene of interest (e.g., nucleotide sequence encoding an antibody) can be placed under the control of any promoter or enhancer element known in the art. Examples of such elements include strong expression promoters (e.g., human CMV IE promoter / enhancer or CMV major IE (CMV-MIE) promoter, as well as RSV, SV40 late promoter, SL3-3, MMTV, ubiquitin (Ubi), ubiquitin C (UbC), and HIV LTR promoter).

[0115] In some embodiments, the vector comprises a promoter selected from the group consisting of SV40, CMV, CMV-IE, CMV-MIE, RSV, SL3-3, MMTV, Ubi, UbC, and HIV LTR.

[0116] The nucleic acid molecules of the present invention may also be operably linked to an effective poly (A) termination sequence, an origin of replication for plasmid products in E. coli, an antibiotic resistance gene as a selectable marker, and / or a convenient cloning site (e.g., a polylinker). The nucleic acid may also include an adjustable inducible promoter (inducible, repressible, developmentally regulated) as opposed to a constitutive promoter, such as CMV IE (the skilled person will recognize that such terms are actually descriptors of gene expression levels under certain conditions).

[0117] Selectable markers are elements well known in the art. In some cases, in addition to GPT, additional selectable markers may be used, wherein such markers make the cells visible. Positive or negative selection may be used.

[0118] In some embodiments, the vector includes one or more selectable marker genes encoding green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), cyan fluorescent protein (CFP), enhanced cyan fluorescent protein (eCFP), yellow fluorescent protein (YFP), or enhanced yellow fluorescent protein (eYFP).

[0119] For purposes of the present invention, gene expression in eukaryotic cells can be tightly regulated using a strong promoter, which is controlled by an operator, and the operator is then regulated by a regulatory fusion protein (RFP). RFP consists essentially of a transcription blocking domain and a ligand binding domain that regulates its activity. Examples of such expression systems are described in US20090162901A1, which is incorporated herein by reference in its entirety.

[0120] Many operons in eukaryotic cells and bacteriophages have been well characterized (Neidhardt, ed., Escherichia coli and Salmonella; Cellular and Molecular Biology, 2nd edition, Vol. 2, ASM Press, Washington D.C. 1996). These include, but are not limited to, the operator region of the LexA gene of E. coli, which is associated with the LexA peptide, and the lactose and tryptophan operons, which are associated with the repressor proteins encoded by the LacI and trpR genes of E. coli. These also include the λP R The phage operon of the phage P22 ant / mnt gene is combined with the repressor protein encoded by λcI and P22 arc. In some embodiments, when the transcription blocking domain of the repressor protein is a restriction enzyme such as NotI, the operon is the recognition sequence of the enzyme. Those skilled in the art will recognize that the operon must be located near the promoter or at the 3' of the promoter so that it can control transcription by the promoter. For example, U.S. Patent No. 5,972,650 (incorporated herein by reference) stipulates that the tetO sequence is within a specific distance of the TATA box. In specific embodiments, the operon is preferably placed just downstream of the promoter. In other embodiments, the operon is placed within 10 base pairs of the promoter.

[0121] In certain embodiments, the operon is selected from the group consisting of: tet operon (tetO), NotI recognition sequence, LexA operon, lactose operon, tryptophan operon and Arc operon (AO). In some embodiments, the repressor protein is selected from TetR, LexA, LacI, TrpR, Arc, λC1 and GAL4. In other embodiments, the transcription blocking domain is derived from a eukaryotic repressor protein, such as a repressor domain derived from GAL4.

[0122] In an exemplary cell expression system, cells are engineered to express tetracycline repressor (TetR) and the protein of interest is placed under the transcriptional control of a promoter whose activity is regulated by TetR. Two tandem TetR operators (tetO) are placed just downstream of the CMV-MIE promoter / enhancer in the vector. The transcription of the gene encoding the protein of interest directed by the CMV-MIE promoter in such a vector can be blocked by TetR in the absence of tetracycline or in the presence of some other suitable inducers (e.g., doxycycline). In the presence of an inducer, the TetR protein can bind to tetO, thereby causing transcription of the protein of interest and then causing translation (expression) to occur (see, e.g., U.S. Pat. No. 7,435,553, which is incorporated herein by reference in its entirety).

[0123] Another exemplary cell expression system includes a regulatory fusion protein such as TetR-ER LBD T2 fusion protein, wherein the transcription blocking domain of the fusion protein is TetR and the ligand binding domain is an estrogen receptor ligand binding domain (ER ) with a T2 mutation LBD )(ER LBD T2; Feil et al. (1997) Biochem. Biophys. Res. Commun. 237: 752-757). When the tetO sequence is placed downstream and near the strong CMV-MIE promoter, transcription of the nucleotide sequence of interest from the CMV-MIE / tetO promoter is blocked in the presence of tamoxifen and unblocked by removing tamoxifen. In another example, the fusion protein Arc2-ER LBD T2 (a single-chain dimer consisting of two Arc proteins connected by a 15-amino acid linker and ER LBD The fusion protein composed of T2 (supra) involves the Arc operator (AO), more specifically, two tandem arc operators just downstream of the CMV-MIE promoter / enhancer. LBD T2 regulation, in which cells expressing the protein of interest are driven by the CMV-MIE / ArcO2 promoter and can be induced by removing tamoxifen. (See, e.g., US20090162901A1, which is incorporated herein by reference).

[0124] In some embodiments, the vectors of the present invention include a CMV-MIE / TetO or CMV-MIE / AO2 hybrid promoter.

[0125] The vectors of the present invention can also use the Cre-lox tool for recombination technology to facilitate the replication of the gene of interest. The Cre-lox strategy requires at least two components: 1) Cre recombinase, an enzyme that catalyzes recombination between two loxP sites; and 2) loxP sites (e.g., a specific 34-base pair bp sequence consisting of an 8-bp core sequence (in which recombination occurs) and two flanking 13-bp inverted repeat sequences) or mutant lox sites. (See, e.g., Araki et al. PNAS 92: 160-4 (1995); Nagy, A. et al. Genesis 26: 99-109 (2000); Araki et al. Nuc Acids Res 30 (19): e103 (2002) and US20100291626A1, all of which are incorporated herein by reference). In another recombination strategy, yeast-derived FLP recombinase can be used together with the consensus sequence FRT (see also, eg, Dymecki, S. PNAS 93(12):6191-6196 (1996)).

[0126] In another aspect, the gene (i.e., the nucleotide sequence encoding the recombinant polypeptide of the present invention) is inserted into the upstream or downstream of the GPT gene of the expression cassette, and is optionally operably connected to a promoter, wherein the 5' flank of the gene connected to the promoter is the first recombinase recognition site and the 3' flank is the second recombinase recognition site. Such recombinase recognition sites allow Cre-mediated recombination in the host cell of the expression system. In some cases, the gene connected to the second promoter is downstream (3') of the first gene and the 3' flank is the second recombinase recognition site. In other cases, the 5' flank of the gene connected to the second promoter is the second recombinase recognition site, and the 3' flank is the third recombinase recognition site. In some embodiments, the recombinase recognition site is selected from loxP site, lox511 site, lox2272 site and FRT site. In other embodiments, the recombinase recognition site is different. In another embodiment, the host cell includes a gene that can express the Cre recombinase.

[0127] In one embodiment, the vector includes a first gene encoding an antibody light chain or an antibody heavy chain of the present invention, and a second gene encoding an antibody light chain or an antibody heavy chain of the present invention.

[0128] In some embodiments, the vector further comprises an X-box binding protein 1 (mXBP1) gene, which can further enhance protein production / protein secretion by controlling the expression of genes involved in protein folding in the endoplasmic reticulum (ER). (See, e.g., Ron D and Walter P. Nat Rev Mol Cell Biol. 8:519–529 (2007)).

[0129] Any cell is suitable for expressing the recombinant nucleic acid sequence of the present invention. Cells used for the present invention include mammalian cells, such as non-human animal cells, human cells or cell fusions, such as hybridomas or quadromas. In certain embodiments, the cell is a human, monkey, hamster, rat or mouse cell. In other embodiments, the cell is a eukaryotic cell and is selected from the following cells: CHO (e.g., CHO K1, DXB-11CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293EBNA, MSR 293, MDCK, HaK, BHK21), HeLa, HepG2, WI38, MRC 5, Colo25, HB 8065, HL-60, Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT cells, tumor cells and cell lines derived from the aforementioned cells. In some embodiments, the cell comprises one or more viral genes, such as a retinal cell expressing a viral gene (e.g., PER.C6 cell).

[0130] In yet another aspect, the present invention relates to recombinant mammalian host cells, such as transfectomas, which produce immunoglobulins, such as antibodies or bispecific molecules. Examples of such host cells include engineered mammalian cells, such as CHO or HEK cells. For example, in one embodiment, the present invention provides cells comprising nucleic acids stably integrated into the cell genome, the nucleic acids comprising sequences encoding antibody expression, and the antibodies comprising recombinant polypeptides of the present invention. In another embodiment, the present invention provides cells comprising non-integrated (i.e., episomal) nucleic acids (such as plasmids, cosmids, phasmids, or linear expression elements), the nucleic acids comprising sequences encoding antibody expression, and the antibodies comprising recombinant polypeptides of the present invention. In other embodiments, the present invention provides cell lines produced by stably transfecting host cells with plasmids comprising expression vectors of the present invention.

[0131] Thus, in one aspect, the invention provides a cell comprising: (a) a recombinant polynucleotide encoding an exogenously added mammalian GPT gene and (b) a polynucleotide encoding a multi-subunit protein. In some embodiments, the exogenously added GPT gene is 90% identical to the nucleic acid sequence of SEQ ID NO: 2, non-limiting examples of which are provided in SEQ ID NO: 11-17, and the multi-subunit protein is an antibody. In other embodiments, the cell further contains an exogenously added GPT gene and a regulatory element. In one embodiment, the cell is a mammalian cell, such as a CHO cell used to prepare a biopharmaceutical.

[0132] In another aspect, the present invention provides a cell line derived from the cells described in the previous aspects. By "derived from" is meant a population of cells that are asexually propagated by individual cells and have certain selected qualities (such as the ability to produce active protein at a given titer, or the ability to proliferate to a specific density). In some embodiments, a cell line derived from a cell having a recombinant polynucleotide encoding a mammalian GPT gene and a polynucleotide encoding a multisubunit protein is capable of producing a multisubunit protein at a titer of at least 3 grams per liter (g / L) of culture medium, at least 5g / L, or at least 8g / L. In some embodiments, the cell line can achieve a cumulative cell density (ICD) that is at least 30%, at least 50%, at least 60%, or at least 90% greater than that achieved by a cell line derived from cells that are substantially identical but do not have a recombinant polynucleotide encoding a GPT.

[0133] Methods for amplifying a GOI are provided. An illustrative method applies increasing concentrations of tunicamycin to a eukaryotic GPT expression system, thereby amplifying gene copies of a GOI operably linked to an exogenously added mammalian GPT gene.

[0134] Protein of interest

[0135] The nucleic acid sequence encoding the gene of interest can be conveniently integrated into a cell comprising a Tn resistance marker gene and an IRES, and optionally flanked by recombinase recognition sites. Any protein of interest suitable for expression in mammalian cells can be used, however, glycoproteins will particularly benefit from the methods of the present invention. For example, the protein of interest can be an antibody or antigen-binding fragment thereof, a bispecific antibody or fragment thereof, a chimeric antibody or fragment thereof, a ScFv or fragment thereof, an Fc tag protein (e.g., a Trap protein) or a fragment thereof, a growth factor or a fragment thereof, a cytokine or a fragment thereof, an extracellular domain of a cell surface receptor or a fragment thereof.

[0136] Glycoproteins with asparagine-linked (N-linked) glycans are ubiquitous in eukaryotic cells. The biosynthesis of these glycans and their conversion to polypeptides occurs in the endoplasmic reticulum (ER). The N-glycan structure is further modified by a number of glycosidases and glycosyltransferases in the ER and Golgi complex. Protein preparation using the present invention is directed to the consistency of the native N-glycan structure to eliminate immunogenic epitopes ("glycotopes").

[0137] Using the method of the present invention, the recombinant protein batches exhibit favorable properties. HPLC (with fluorescence detection) of repeated protein production batches proves that the glycoprotein has a uniform expression and glycosylation pattern, as illustrated in Figures 7-8 herein. A method for glycosylation of an N-glycan protein substrate is provided, wherein a mammalian host cell encoding a nucleic acid molecule is provided, the nucleic acid molecule comprising a mammalian tunicamycin (Tn) resistance gene operably linked to a gene encoding a protein substrate that needs glycosylation; the cell is cultured in the presence of a first concentration of Tn; a cell population expressing at least one copy of the Tn resistance gene is separated; the cell population is cultured in the presence of an increasing concentration of Tn; and the N-glycan protein substrate is separated from the cell culture. The N-glycan content of the protein substrate can be evaluated by any method known in the art for the presence of monosaccharides and oligosaccharides.

[0138] Detailed structural analysis of glycan-attached proteins can be correlated with the functional characteristics of the proteins. Such analysis to characterize protein glycosylation typically involves several steps: i) enzymatic or chemical release of attached glycans; ii) derivatization of released glycans via reductive amination or permethylation of aromatic or aliphatic amines; iii) analysis of glycans. Many variations of analyzing glycosylation patterns are known to the skilled artisan. Glycoproteins can carry several forms of glycoforms occupying each site in specific numbers, and therefore, their complexity may make them difficult to reproduce in certain production methods. The consistency of the type and number of glycoforms is measurable and represents a desired outcome for therapeutic protein production.

[0139] Host cells and transfection

[0140] The mammalian host cell used in the method of the present invention is a eukaryotic host cell, typically a mammalian cell, including, for example, a CHO cell and a mouse cell. In one embodiment, the present invention provides a cell comprising a nucleic acid sequence (as shown in SEQ ID NO:3) encoding a Tn resistance marker protein derived from a Chinese hamster (Cricetulus griseus) or a homologue or variant thereof. In some embodiments, the cell includes multiple gene copies of a Tn resistance marker gene. In other embodiments, the present invention provides a nucleic acid sequence encoding a Tn resistance marker protein derived from humans (SEQ ID NO:4), macaques (SEQ ID NO:5), chimpanzees (SEQ ID NO:6), dogs (SEQ ID NO:7), guinea pigs (SEQ ID NO:8), rats (SEQ ID NO:9) or mice (SEQ ID NO:10).

[0141] The present invention includes mammalian host cells transfected with the expression vector of the present invention. Transfected host cells include cells transfected with expression vectors, and the expression vectors include sequences encoding proteins or polypeptides of interest. The expressed protein will usually be secreted into the culture medium depending on the selected nucleic acid sequence, but may remain in the cell or be deposited in the cell membrane. Various mammalian cell culture systems can be used to express recombinant proteins. Examples of suitable mammalian host cell lines include the COS-7 cell line of monkey kidney cells described in Gluzman (1981) Cell 23:175, and other cell lines capable of expressing appropriate vectors, including, for example, CV-1 / EBNA (ATCC CRL 10478), L cells, C127, 3T3, CHO, HeLa and BHK cell lines. Other cell lines developed for specific selection or amplification processes can also be used together with the methods and compositions provided herein. In one embodiment of the present invention, the cell is a CHO cell line called K1 (i.e., CHO K1 cell). In order to achieve the goal of high yield of recombinant protein, the host cell line should be pre-adapted to the bioreactor culture medium under appropriate circumstances.

[0142] Several transfection methods are known in the art and are reviewed in Kaufman (1988) Meth. Enzymology 185:537. The selected transfection protocol will depend on the host cell type and the nature of the GOI and can be selected based on routine experiments. The basic requirement of any such protocol is to first introduce the DNA encoding the protein of interest into a suitable host cell and then identify and isolate the host cell that has contained the heterologous DNA in a relatively stable, expressible manner.

[0143] Some reagents that can be used to introduce heterologous DNA into mammalian cells include Lipofectin TM Reagents and Lipofectamine TM Reagents (Gibco BRL, Gaithersburg, Md.). Both reagents are commercially available reagents for forming lipid-nucleic acid complexes (or liposomes) that, when applied to cultured cells, facilitate the uptake of nucleic acids into the cells.

[0144] The selected transfection scheme and the elements selected for use therein will depend on the type of host cell used. Those skilled in the art know many different schemes and host cells, and can select the appropriate system for expressing the desired protein based on the requirements of the cell culture system used. In another aspect, the present invention relates to expression vectors encoding polypeptides, including but not limited to antibodies, bispecific antibodies, chimeric antibodies, ScFv, antigen binding proteins or Fc fusion proteins. Such expression vectors can be used for the preparation of recombinant polypeptides using the method and composition of the present invention.

[0145] Other features of the present invention will become apparent in the course of the following description of exemplary embodiments, which are intended to illustrate the invention and are not intended to limit the invention.

[0146] Example

[0147] The following examples are provided to describe to those of ordinary skill in the art how to construct and use the methods and compositions of the present invention and are not intended to limit the scope of what the inventors consider to be their invention. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but certain experimental errors and deviations should be considered. Unless otherwise indicated, parts are parts by weight, molecular weights are average molecular weights, temperatures are degrees Celsius, and pressures are at or near atmospheric pressure.

[0148] Example 1 Selection efficiency of transfected cells expressing GPT.

[0149] The modified CHO K1 cells are transfected with a plasmid vector containing CHO-GPT (SEQ ID NO: 2), human GPT (SEQ ID NO: 12) or a plasmid vector containing hygromycin phosphotransferase (Hpt, hygromycin resistance gene); for example, the selectable marker gene (CHO-GPT or hpt) is transcriptionally linked to the downstream eGFP gene via an IRES sequence in its corresponding vector. For example, each plasmid is constructed to include the following gene sequences in the 5' to 3' direction: Lox site, SV40 late promoter, CHO-GPT (or Hpt), IRES, enhanced green fluorescent protein (eGFP) and a second Lox site. The purified recombinant plasmid is co-transfected with a plasmid expressing the Cre recombinase into a modified CHO host cell containing the following sites at the transcriptionally active locus from 5' to 3': lox site, YFP and a second lox site. Therefore, the host CHO cells can be separated as green positive or yellow negative cells by flow cytometry. When a recombinant plasmid expressing eGFP (transcriptionally regulated by GPT or hpt gene) is co-transfected with a plasmid expressing Cre recombinase, the recombination mediated by Cre recombinase leads to site-specific integration of the GPT / eGFP cassette at the chromosomal locus containing lox sites, and replacement of the YFP gene occurs (i.e., green positive cells). If eGFP is randomly integrated, both green positive and yellow positive cells will be produced.

[0150] The cell populations were incubated with 0, 1 μg / ml, 2.5 μg / ml or 5 μg / ml tunicamycin (Tn) or 400 μg hygromycin (Hyg) as summarized in Table 2. The observed recombinant population (ORP) was measured by fluorescence activated cell sorting (FACS) analysis. The cells were sorted to quantify each cell population and the selection efficiency was calculated for cells expressing only GFP but not YFP (Figure 4 or 5).

[0151] The selection efficiency (percentage of the population of surviving cells expressing GFP) was compared between pools of cells resistant to Tn or Hyg (Table 2).

[0152] Table 2: Selection efficiency

[0153]

[0154] It was observed that tunicamycin selection was as efficient as hygromycin selection. Both CHO-GPT and human GPT were efficient in selecting integrants in the presence of either 1 μg / ml or 2.5 μg / ml tunicamycin.

[0155] Example 2 Amplification of gene products.

[0156] Incremental selection is performed by applying increasing concentrations of tunicamycin to the GPT expression system. CHO K1 cells are transfected with a plasmid vector containing the CHO-GPT gene (SEQ ID NO: 2) as above. The plasmid contains a first Lox site, an SV40 late promoter, a CHO-GPT gene, an IRES, eGFP, and a second Lox site in a 5' to 3' direction. The CRE-lox site guides the integration of the gene of interest into the genome, thereby producing a stable transfected cell pool with at least one GPT insertion per cell. (As seen above, more integrants can be produced due to random integration). CHO cells were initially cultured in the presence of 1 μg / ml tunicamycin (Tn). Transfectants were then selected from a stable pool (referred to as cell pool 2) and subsequently amplified in the presence of 1 μg / ml, 2.5 μg / ml, or 5 μg / ml Tn. Several rounds of selection were performed to identify cell populations that can enhance eGFP expression (multiple copies). In the presence of 2.5 μg / ml or 5 μg / ml Tn, random integration events were greatly increased.

[0157] The copy number of the gene product CHO GPT, eGFP or mGapdh (normalization control) was measured using standard qPCR methods. The eGFP copy number in cells from the 1 μg / ml Tn-resistant pool that was further incubated with 2.5 μg / ml Tn was at least 2 times the eGFP copy number from the 1 μg / ml Tn-resistant pool that was further incubated with 1 μg / ml Tn. When the pool treated with 1 μg / ml Tn was further incubated with 5 μg / ml Tn, the gene copy number increased further. The increase in eGFP gene copy number correlated with the increased CHO-GPT gene copies. (See Fig. 6A and 6B ).

[0158] To determine whether the increase in gene copy number would translate into increased protein expression, mean fluorescence intensity (MFI) was measured by FACS for the same pool of cells expressing GPT and eGFP that had been treated with multiple rounds of Tn selection (i.e., 1, 2.5, or 5 μg Tn) (see e.g. Figure 6B 7, 8 and 9 in ). A comparison of eGFP expression of these cell pools is shown in Table 3.

[0159] In terms of eGFP production, the GPT-expressing cell pool subjected to a second round of 5 μg Tn selection had exactly 2.5-fold higher yields compared to the 1 μg Tn treatment and 1.5-fold higher yields compared to the 2.5 μg Tn treatment (Table 3).

[0160] Table 3: eGFP protein production

[0161] GPT 1μg pool + second Tn (μg) treatment MFI 1 μg 1098 2.5 μg 1867 5μg 2854

[0162] Without being bound by any one theory, increasing the concentration of Tn amplifies the selective pressure on the cells in a controlled manner, thereby increasing yield.

[0163] As described below, the Tn-resistant expression vector was also used in additional experiments to examine the effect of Tn selection on glycosylation patterns.

[0164] Example 3 Expression and glycosylation profile of exemplary dimeric proteins.

[0165] CHO cells expressing "Trap" protein (Fc fusion protein 1, hereinafter referred to as FcFP1) were transfected with an expression vector containing GPT. The plasmid has a Lox site, SV40 late promoter, Tn resistance gene (CHO-GPT), IRESeGFP, SV40 polyA and a second Lox site in the 5' to 3' direction. 1 μg / mL Tn or 5 μg / mL Tn was used to select the GPT selective marker. The selected cell pool was amplified by suspension culture in a serum-free production medium. GPT transfection was confirmed by the expression of eGFP by FACS analysis. The mass collected from the selected pool was sent for copy number analysis of GPT expression, and a 12-day productivity analysis was established to determine the FcFP1 expression level in the pool selected with different concentrations of tunicamycin.

[0166] FcFP1 was selected for its complex glycosylation pattern, which has abundant glycosylation sites. To determine the glycosylation profile, cells expressing FcF1 protein were expanded by cell culture under standard protocols (no Tn) or Tn-treated conditions (as shown in Table 4), and the protein was then isolated and purified.

[0167] Table 4: FcFP1 protein production

[0168] protein batch number deal with FcFP1 Trap 110728 none FcFP1 Trap 110728-1 1 μg / ml Tn FcFP1 Trap 110728-2 5 μg / ml Tn

[0169] Detailed glycan analysis was performed on each batch of glycoprotein using chromatography based on the well-known HPLC method and fluorescent anthranilic acid (AA) label (Anumula and Dhume, Glycobiology, 8(7):685-694, 1998) to determine whether Tn had a negative impact on the glycosylation profile. Production batches were also compared to reference standards representing therapeutically acceptable protein batches. Representative glycan analysis is shown in Figure 2. Figures 7A-7D Each batch (compared to the reference batch) consistently produced the same number of peaks, relative shapes, and relative intensities. The overlay of each chromatogram ( Figure 8 ) shows that there are no unique or unusual peaks.

[0170] Oligosaccharide properties of FcFP1 protein were analyzed by well-known HPLC methods for reference standard batches. The amount of sialylation was measured for FcFP1 trap protein batches, and the Z number was calculated for each batch (3 replicates). The Z number represents a measure of the variation between batches. The Z number takes into account the relative number of peaks and the relative shape and intensity of each peak. For example, Figures 7A-7D The areas of the 0SA, 1SA, 2SA, 3SA and 4SA peaks are quantified as shown in Table 5.

[0171] Table 5: Oligosaccharide Quantification Analysis

[0172]

[0173] OS = oligosaccharide; OSA = 0 sialic acid residues; 1SA = 1 sialic acid residue; 2SA = 2 sialic acid residues; 3SA = 3 sialic acid residues; 4SA = 4 sialic acid residues

[0174]

[0175] The calculated Z numbers for each batch were within acceptable ranges compared to the reference batch, so it is understood that each protein batch achieved the same material as the therapeutic molecule. Since the presence of Tn is known to have a negative effect on the glycosylation of N-linked glycoproteins, it was unexpected that protein production and yields were reliable and consistent given the increased selective pressure conditions created by Tn.

[0176] The present invention may be embodied in other specific embodiments without departing from the spirit or substance of the invention.

[0177] In summary, this application relates to the following implementation schemes:

[0178] 1. An isolated cell comprising a mammalian tunicamycin (Tn) resistance gene encoding a protein having at least 93% identity to the amino acid sequence of SEQ ID NO: 3, said gene being operably linked to a gene of interest (GOI) and at least one regulatory element.

[0179] 2. The isolated cell according to embodiment 1, wherein the Tn resistance gene is exogenously added to the cell.

[0180] 3. The isolated cell according to embodiment 1 or 2, wherein the Tn resistance gene encodes a protein having at least 95% identity with the amino acid sequence of SEQ ID NO: 3.

[0181] 4. The isolated cell according to any one of embodiments 1 to 3, wherein the Tn resistance gene encodes a protein having at least 98% identity to the amino acid sequence of SEQ ID NO: 3.

[0182] 5. The isolated cell of embodiment 1 or 2, wherein the Tn resistance gene comprises a nucleic acid sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17.

[0183] 6. The isolated cell of embodiment 1 or 2, wherein the Tn resistance gene comprises a nucleic acid sequence comprising SEQ ID NO: 12.

[0184] 7. An isolated cell according to any one of embodiments 1 to 6, wherein the at least one regulatory element is selected from a promoter, a ribosome binding site and an enhancer.

[0185] 8. The isolated cell according to any one of embodiments 1 to 7, further comprising a second gene of interest (GOI).

[0186] 9. The isolated cell according to any one of embodiments 1 to 8, wherein the gene of interest (GOI) is an exogenously added GOI.

[0187] 10. The isolated cell according to any one of embodiments 1 to 9, wherein the exogenously added GOI is a human gene.

[0188] 11. The isolated cell according to any one of embodiments 1 to 10, wherein the regulatory element is an exogenously added regulatory element.

[0189] 12. The isolated cell according to any one of embodiments 1 to 11, wherein the first GOI and / or the second GOI is selected from an antibody heavy chain, an antibody light chain, an antigen binding fragment and an Fc fusion protein.

[0190] 13. The isolated cell according to any one of embodiments 8 to 12, wherein the first GOI and the second GOI are independently selected from genes encoding antibody light chains or antigen-specific fragments thereof, antibody heavy chains or antigen-specific fragments thereof, Fc fusion proteins or fragments thereof, and receptors or ligand-specific fragments thereof.

[0191] 14. The isolated cell of any one of embodiments 1 to 113, wherein the GOI is operably linked to a promoter.

[0192] 15. The isolated cell of any one of embodiments 1 to 14, wherein the GOI encodes a glycoprotein selected from an antibody light chain or an antigen-binding fragment thereof, an antibody heavy chain or an antigen-binding fragment thereof, an Fc fusion protein or a fragment thereof, a ligand, and a receptor or a ligand-binding fragment thereof.

[0193] 16. The isolated cell according to any one of embodiments 8 to 15, wherein a recombinase recognition site is present between the first GOI and the second GOI.

[0194] 17. The isolated cell of embodiment 8, further comprising a recombinase recognition site 5' to the first GOI and a recombinase recognition site 3' relative to the second GOI.

[0195] 18. The isolated cell of any one of embodiments 1 to 17, wherein the cell is a eukaryotic cell.

[0196] 19. The isolated cell of any one of embodiments 1 to 18, wherein the cell is a mammalian cell.

[0197] 20. The isolated cell according to any one of embodiments 1 to 18, wherein the cell is selected from CHO-K1, COS-7, HEK293, a tumor cell, a lymphocyte, a retinal cell, and a stem cell.

[0198] 21. The isolated cell according to any one of embodiments 1 to 20, wherein the cell is a CHO-K1 cell.

[0199] 22. A method for preparing a recombinant protein of interest (POI), wherein the method comprises:

[0200] a. providing a mammalian host cell encoding a nucleic acid molecule, the cell comprising (i) a mammalian tunicamycin (Tn) resistance gene and (ii) a gene encoding the POI;

[0201] b. culturing the cells in the presence of a first concentration of Tn;

[0202] c. isolating a population of cells expressing at least one copy of the Tn resistance gene;

[0203] d. culturing the cell population in the presence of increasing concentrations of Tn, wherein increasing the concentration of Tn increases production of the POI; and

[0204] e. Isolating the POI from cell culture.

[0205] 23. The method of embodiment 22, wherein the mammalian Tn resistance gene comprises a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 2.

[0206] 24. The method of embodiment 22, wherein the mammalian Tn resistance gene comprises a nucleic acid sequence that is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 2.

[0207] 25. The method of any one of embodiments 22 to 24, wherein the mammalian Tn resistance gene comprises a Chinese hamster (Cricetulus griseus) Tn resistance gene.

[0208] 26. The method of embodiment 22, wherein the mammalian Tn resistance gene comprises a human Tn resistance gene.

[0209] 27. The method of embodiment 22, wherein the mammalian Tn resistance gene comprises a nucleic acid sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17.

[0210] 28. The method of any one of embodiments 22 to 27, wherein the Tn resistance gene is operably linked to the gene encoding the POI.

[0211] 29. The method according to any one of embodiments 22 to 27, wherein the gene encoding the POI is operably linked to at least one regulatory element.

[0212] 30. The method of embodiment 29, wherein the at least one regulatory element is selected from a promoter, a ribosome binding site, and an enhancer.

[0213] 31. The method of any one of embodiments 22 to 30, further comprising a second gene encoding a POI.

[0214] 32. The method of any one of embodiments 22 to 31, wherein the gene is an exogenously added gene of interest (GOI).

[0215] 33. The method of embodiment 32, wherein the exogenously added GOI is a human gene.

[0216] 34. A method according to embodiment 29 or 30, wherein the regulatory element is an exogenously added regulatory element.

[0217] 35. The method of any one of embodiments 22 to 34, wherein the POI is selected from an antibody heavy chain, an antibody light chain, an antigen binding fragment, and an Fc fusion protein.

[0218] 36. The method of any one of embodiments 22 to 35, wherein the first concentration of Tn is 1 μg / mL.

[0219] 37. The method of any one of embodiments 22 to 36, wherein the increasing concentration of Tn comprises a second and a third concentration of Tn.

[0220] 38. A method according to embodiment 37, wherein the second concentration is greater than the Tn of the first concentration, and the third concentration is greater than the Tn of the second concentration.

[0221] 39. The method of embodiment 37 or 38, wherein the second concentration of Tn is 2.5 μg / ml and the third concentration is 5 μg / mL.

[0222] 40. The method of any one of embodiments 22 to 36, wherein the increased concentration of Tn comprises a second concentration of Tn, wherein the second concentration of Tn is 2.5 μg / ml or 5 μg / mL.

[0223] 41. A method for glycosylation of an N-glycan protein substrate, wherein the method comprises:

[0224] a. providing a mammalian host cell encoding a nucleic acid molecule comprising a mammalian tunicamycin (Tn) resistance gene operably linked to a gene encoding a protein substrate requiring glycosylation;

[0225] b. culturing the cells in the presence of a first concentration of Tn;

[0226] c. isolating a population of cells expressing at least one copy of the Tn resistance gene;

[0227] d. culturing the cell population in the presence of increasing concentrations of Tn, wherein increasing the concentration of Tn increases production of the POI; and

[0228] e. isolating the protein substrate from the cell culture.

[0229] 42. The method of embodiment 41, wherein the mammalian Tn resistance gene comprises a nucleic acid sequence that is at least 93% identical to the nucleic acid sequence of SEQ ID NO: 2.

[0230] 43. The method of embodiment 39, wherein the mammalian Tn resistance gene comprises a nucleic acid sequence that is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 2.

[0231] 44. The method of any one of embodiments 41 to 43, wherein the mammalian Tn resistance gene comprises a Chinese hamster (Cricetulus griseus) Tn resistance gene.

[0232] 45. The method of embodiment 41, wherein the mammalian Tn resistance gene comprises a human Tn resistance gene.

[0233] 46. ​​The method of embodiment 41, wherein the mammalian Tn resistance gene comprises a nucleic acid sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17.

[0234] 47. The method of any one of embodiments 41 to 46, wherein the Tn resistance gene is operably linked to the gene encoding the POI.

[0235] 48. The method of any one of embodiments 41 to 47, wherein the gene encoding the POI is operably linked to at least one regulatory element.

[0236] 49. A method according to embodiment 48, wherein the at least one regulatory element is selected from a promoter, a ribosome binding site and an enhancer.

[0237] 50. The method of any one of embodiments 41 to 47, further comprising a second gene of interest (GOI).

[0238] 51. The method of embodiment 50, wherein the gene of interest (GOI) is an exogenously added GOI.

[0239] 52. The method of embodiment 51, wherein the exogenously added GOI is a human gene.

[0240] 53. A method according to embodiment 48 or 49, wherein the regulatory element is an exogenously added regulatory element.

[0241] 54. The method of any one of embodiments 41 to 53, wherein the POI is selected from an antibody heavy chain, an antibody light chain, an antigen binding fragment, and an Fc fusion protein.

[0242] 55. The method of any one of embodiments 41 to 54, wherein the first concentration of Tn is 1 μg / mL.

[0243] 56. The method of any one of embodiments 41 to 55, wherein the increasing concentration of Tn comprises a second and a third concentration of Tn.

[0244] 57. A method according to embodiment 56, wherein the second concentration is greater than the Tn of the first concentration, and the third concentration is greater than the Tn of the second concentration.

[0245] 58. The method of embodiment 56 or 57, wherein the second concentration of Tn is 2.5 μg / ml and the third concentration is 5 μg / mL.

[0246] 59. The method of any one of embodiments 41 to 55, wherein the increased concentration of Tn comprises a second concentration of Tn, wherein the second concentration of Tn is 2.5 μg / ml or 5 μg / mL. Sequence Listing <110> Razerne Pharmaceuticals <120> High efficiency and selectivity of recombinant proteins <130> IIC210883 <150> US 62 / 039,416 <151> 2014-08-19 <160> 17 <170> PatentIn Version 3.5 <210> 1 <211> 6964 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 1 aagcttatac tcgagctcta gattgggaac ccgggtctct cgaattcgag atctagttta 60 aacacgcggc cgctaatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac 120 ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg 180 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa 240 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat 300 gtctaccggt ataacttcgt ataatgtata ctatacgaag ttagccggta gggcccctct 360 cttcatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 420 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 480 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 540 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 600 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg 660 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 720 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 780 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 840 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt 900 taccttcgga aaaagagtg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 960 tggtttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 1020 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 1080 ggtcatgggc gcgcctcata ctcctgcagg catgagatta tcaaaaagga tcttcaccta 1140 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg 1200 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg 1260 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc 1320 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc 1380 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc 1440 ctccatccag tctattaatt gttgccggga agctaggc agtagttcgc cagttaag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat 1560 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt 1680 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatact gcgccacata gcagaacttt aaaagtgctc atcattgga aacgtttttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaac aggaggca aatgccgca aaaagggat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcaggt acacaacttc 2220 gtatagcata cattatacga agttatggta ccaagcctag gcctccaaaa aagcctcctc 2280 actacttctg gaatagctca gaggcagagg cggcctcggc ctctgcataa ataaaaaaaa 2340 ttagtcagcc atggggcgga gaatgggcgg aactgggcgg agttaggggc gggatgggcg 2400 gagttagggg cgggactatg gttgctgact aattgagatg catgctttgc atacttctgc 2460 ctgctgggga gcctggggac tttccacacc tggttgctga ctaattgaga tgcatgcttt 2520 gcatacttct gcctgctggg gagcctgggg actttccaca ccggatccac catgtgggcc 2580 ttcccggagt tgccgctgcc gctgctggtg aatttgttcg gctcgctgct gggatttgtg 2640 gctactgtga ccctcatccc tgccttccgt agccacttta tcgccgcgcg cctctgtggc 2700 caggacctca acaagctcag ccggcagcag atcccagaat cccagggagt gatctgcggt 2760 gctgttttcc ttatcatcct cttctgcttc atccctttcc ccttcctgaa ctgctttgtg 2820 gaggagcagt gtaaggcatt cccccaccat gaatttgtgg ccctgatagg tgccctcctt 2880 gccatctgct gcatgatctt cctgggcttc gctgatgatg tactcaatct gcgctggcgc 2940 cataagctgc tgctgcccac agctgcctct ctacctctcc tcatggttta cttcactaac 3000 tttggcaata caaccattgt ggtacccaag cccttccgct ggattcttgg cctgcatttg 3060 gacttgggaa tcctatacta tgtctacatg ggactgcttg cggtgttctg taccaatgcc 3120 atcaacatcc tagcaggaat taatggccta gaggctggtc agtcactagt catctctgct 3180 tctatcattg tcttcaacct ggtagagctg gaaggtgatt atcgggatga tcatgtcttt 3240 tccctctact tcatgatacc attttttttt accaccttgg gattgctata ccataactgg 3300 tacccatcac aggtgtttgt gggagatacc ttctgttatt ttgctggcat gacctttgcc 3360 gtggtgggaa tcttgggaca cttcagcaag accatgctac tcttctttat tccacaagtg 3420 ttcaatttcc tctactcgct gcctcagctc cttcacgcca tcccctgccc tcgacaccgc 3480 atacccagac tcaatccgaa gacgggcaaa ctggagatga gctattccaa gttcaagacc 3540 aagaacctct ctttcttggg cacctttatt ttaaaggtag cagagcgcct ccagctagtg 3600 acagttcacc gaggcgagag tgaggatggt gccttcactg aatgtaacaa catgaccctc 3660 atcaacttgc tactcaaaat ctttgggccc atacatgaga gaaacctcac actgctcctg 3720 ctgctttgc agatcctgag cagcgctgtc accttctcca ttcgatacca gcttgtccga 3780 ctcttctatg atgtctgaac gcgtcccccc tctccctccc cccccctaa cgttactggc 3840 cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgttattttc caccatattg 3900 ccgtctttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct 3960 aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca 4020 gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg 4080 aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct 4140 gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa 4200 tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt accccattgt 4260 atgggatctg atctggggcc tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa 4320 aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaaaca cgattgctcg 4380 aatcaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga 4440 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc 4500 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg 4560 gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca 4620 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac 4680 catcttcttc areacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga 4740 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct 4800 ggggcacaag ctggagtaca actacaacag ccacaacgtc tacatcatgg ccgacaagca 4860 gaaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca 4920 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga 4980 caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca 5040 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta 5100 caagtaatcg gccgctaatc agccatacca catttgtaga ggttttactt gctttaaaaa 5160 acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact 5220 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata 5280 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc 5340 atgtcggcgc gttgacattg attattgact agttattaat agtaatcaat tacggggtca 5400 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct 5460 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta 5520 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac 5580 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt 5640 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag 5700 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat 5760 gggcgtggat agcggttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat 5820 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc 5880 cattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc 5940 cctatcagtg atagagatct ccctatcagt gatagagatc gtcgacgttt agtgaaccgt 6000 cagatcgcct ggagacgcca tccacgctgt ttgacctcc atagaagaca ccgggaccga 6060 tccagcctcc gcggccggga acggtgcatt ggaacgcgga ttccccgtgc caagagtgac 6120 gtaagtaccg cctatagagt ctataggccc acccccttgg cttcttatgc atgctatact 6180 gttttggct tggggtctat acaccccgc ttcctcatgt tataggtgat ggtatagctt 6240 agcctatagg tgtgggttat tgaccattat tgaccactcc cctattggtg acgatacttt 6300 ccattactaa tccataacat ggctctttgc cacaactctc tttattggct atatgccaat 6360 acactgtcct tcagagactg acacggactc tgtattttta caggatgggg tctcatttat 6420 tatttacaaa ttcacatata caacaccacc gtccccagtg cccgcagttt ttattaaaca 6480 taacgtggga tctccacgcg aatctcgggt acgtgttccg gacatgggct cttctccggt 6540 agcggcggag cttctacatc cgagccctgc tcccatgcct ccagcgactc atggtcgctc 6600 ggcagctcct tgctcctaac agtggaggcc agacttaggc acagcacgat gcccaccacc 6660 accagtgtgc cgcacaaggc cgtggcggta gggtatgtgt ctgaaaatga gctcggggag 6720 cgggcttgca ccgctgacgc atttggaaga cttaaggcag cggcagaaga agatgcaggc 6780 agctgagttg ttgtgttctg ataagagtca gaggtaactc ccgttgcggt gctgttaacg 6840 gtggagggca gtgtagtctg agcagtactc gttgctgccg cgcgcgccac cagacataat 6900 agctgacaga ctaacagact gttcctttcc atgggtcttt tctgcagtca ccgtccttga 6960 cacg 6964 <210> 2 <211> 1231 <212> DNA <213> Chinese hamster <400> 2 caccatgtgg gccttcccgg agttgccgct gccgctgctg gtgaatttgt tcggctcgct 60 gctgggattt gtggctactg tgaccctcat ccctgccttc cgtagccact ttatcgccgc 120 gcgcctctgt ggccaggacc tcaacaagct cagccggcag cagatcccag aatcccaggg 180 agtgatctgc ggtgctgttt tccttatcat cctcttctgc ttcatccct tccccttcct 240 gaactgcttt gtggaggagc agtgtaaggc attcccccac catgaatttg tggccctgat 300 aggtgccctc cttgccatct gctgcatgat cttcctgggc ttcgctgatg atgtactcaa 360 tctgcgctgg cgccataagc tgctgctgcc cacagctgcc tctctacctc tcctcatggt 420 ttacttcact aactttggca atacaaccat tgtggtaccc aagcccttcc gctggattct 480 tggcctgcat ttggacttgg gaatcctata ctatgtctac atgggactgc ttgcggtgtt 540 ctgtaccaat gccatcaaca tcctagcagg aattaatggc ctagaggctg gtcagtcact 600 agtcatctct gcttctatca ttgtcttcaa cctggtagag ctggaaggtg attatcggga 660 tgatcatgtc ttttccctct acttcatgat accatttttt tttaccacct tgggattgct 720 ataccataac tggtacccat cacaggtgtt tgtgggagat accttctgtt atttgctgg 780 catgaccttt gccgtggtgg gaatcttggg acacttcagc aagaccatgc tactcttctt 840 tattccacaa gtgttcaatt tcctctactc gctgcctcag ctccttcacg ccatcccctg 900 ccctcgacac cgcataccca gactcaatcc gaagacgggc aaactggaga tgagctattc 960 caagttcaag accaagaacc tctctttctt gggcaccttt attttaaagg tagcagagcg 1020 cctccagcta gtgacagttc accgaggcga gagtgaggat ggtgccttca ctgaatgtaa 1080 caacatgacc ctcatcaact tgctactcaa aatctttggg cccatacatg agagaaacct 1140 cacactgctc ctgctgcttt tgcagatcct gagcagcgct gtcaccttct ccattcgata 1200 ccagcttgtc cgactcttct atgatgtctg a 1231 <210> 3 <211> 408 <212> PRT <213> Chinese hamster <400> 3 Met Trp Ala Phe Pro Glu Leu Pro Leu Pro Leu Leu Val Asn Leu Phe 1 5 10 15 Gly Ser Leu Leu Gly Phe Val Ala Thr Val Thr Leu Ile Pro Ala Phe 20 25 30 Arg Ser His Phe Ile Ala Ala Arg Leu Cys Gly Gln Asp Leu Asn Lys 35 40 45 Leu Ser Arg Gln Gln Ile Pro Glu Ser Gln Gly Val Ile Cys Gly Ala 50 55 60 Val Phe Leu Ile Ile Leu Phe Cys Phe Ile Pro Phe Pro Phe Leu Asn 65 70 75 80 Cys Phe Val Glu Glu Gln Cys Lys Ala Phe Pro His His Glu Phe Val 85 90 95 Ala Leu Ile Gly Ala Leu Leu Ala Ile Cys Cys Met Ile Phe Leu Gly 100 105 110 Phe Ala Asp Asp Val Leu Asn Leu Arg Trp Arg His Lys Leu Leu Leu 115 120 125 Pro Thr Ala Ala Ser Leu Pro Leu Leu Met Val Tyr Phe Thr Asn Phe 130 135 140 Gly Asn Thr Thr Ile Val Val Pro Lys Pro Phe Arg Trp Ile Leu Gly 145 150 155 160 Leu His Leu Asp Leu Gly Ile Leu Tyr Tyr Val Tyr Met Gly Leu Leu 165 170 175 Ala Val Phe Cys Thr Asn Ala Ile Asn Ile Leu Ala Gly Ile Asn Gly 180 185 190 Leu Glu Ala Gly Gln Ser Leu Val Ile Ser Ala Ser Ile Ile Val Phe 195 200 205 Asn Leu Val Glu Leu Glu Gly Asp Tyr Arg Asp Asp His Val Phe Ser 210 215 220 Leu Tyr Phe Met Ile Pro Phe Phe Phe Thr Thr Leu Gly Leu Leu Tyr 225 230 235 240 His Asn Trp Tyr Pro Ser Gln Val Phe Val Gly Asp Thr Phe Cys Tyr 245 250 255 Phe Ala Gly Met Thr Phe Ala Val Val Gly Ile Leu Gly His Phe Ser 260 265 270 Lys Thr Met Leu Leu Phe Phe Ile Pro Gln Val Phe Asn Phe Leu Tyr 275 280 285 Ser Leu Pro Gln Leu Leu His Ala Ile Pro Cys Pro Arg His Arg Ile 290 295 300 Pro Arg Leu Asn Pro Lys Thr Gly Lys Leu Glu Met Ser Tyr Ser Lys 305 310 315 320 Phe Lys Thr Lys Asn Leu Ser Phe Leu Gly Thr Phe Ile Leu Lys Val 325 330 335 Ala Glu Arg Leu Gln Leu Val Thr Val His Arg Gly Glu Ser Glu Asp 340 345 350 Gly Ala Phe Thr Glu Cys Asn Asn Met Thr Leu Ile Asn Leu Leu Leu 355 360 365 Lys Ile Phe Gly Pro Ile His Glu Arg Asn Leu Thr Leu Leu Leu Leu 370 375 380 Leu Leu Gln Ile Leu Ser Ser Ala Val Thr Phe Ser Ile Arg Tyr Gln 385 390 395 400 Leu Val Arg Leu Phe Tyr Asp Val 405 <210> 4 <211> 408 <212> PRT <213> Homo sapiens <400> 4 Met Trp Ala Phe Ser Glu Leu Pro Met Pro Leu Leu Ile Asn Leu Ile 1 5 10 15 Val Ser Leu Leu Gly Phe Val Ala Thr Val Thr Leu Ile Pro Ala Phe 20 25 30 Arg Gly His Phe Ile Ala Ala Arg Leu Cys Gly Gln Asp Leu Asn Lys 35 40 45 Thr Ser Arg Gln Gln Ile Pro Glu Ser Gln Gly Val Ile Ser Gly Ala 50 55 60 Val Phe Leu Ile Ile Leu Phe Cys Phe Ile Pro Phe Pro Phe Leu Asn 65 70 75 80 Cys Phe Val Lys Glu Gln Cys Lys Ala Phe Pro His His Glu Phe Val 85 90 95 Ala Leu Ile Gly Ala Leu Leu Ala Ile Cys Cys Met Ile Phe Leu Gly 100 105 110 Phe Ala Asp Asp Val Leu Asn Leu Arg Trp Arg His Lys Leu Leu Leu 115 120 125 Pro Thr Ala Ala Ser Leu Pro Leu Leu Met Val Tyr Phe Thr Asn Phe 130 135 140 Gly Asn Thr Thr Ile Val Val Pro Lys Pro Phe Arg Pro Ile Leu Gly 145 150 155 160 Leu His Leu Asp Leu Gly Ile Leu Tyr Tyr Val Tyr Met Gly Leu Leu 165 170 175 Ala Val Phe Cys Thr Asn Ala Ile Asn Ile Leu Ala Gly Ile Asn Gly 180 185 190 Leu Glu Ala Gly Gln Ser Leu Val Ile Ser Ala Ser Ile Ile Val Phe 195 200 205 Asn Leu Val Glu Leu Glu Gly Asp Cys Arg Asp Asp His Val Phe Ser 210 215 220 Leu Tyr Phe Met Ile Pro Phe Phe Phe Thr Thr Leu Gly Leu Leu Tyr 225 230 235 240 His Asn Trp Tyr Pro Ser Arg Val Phe Val Gly Asp Thr Phe Cys Tyr 245 250 255 Phe Ala Gly Met Thr Phe Ala Val Val Gly Ile Leu Gly His Phe Ser 260 265 270 Lys Thr Met Leu Leu Phe Phe Met Pro Gln Val Phe Asn Phe Leu Tyr 275 280 285 Ser Leu Pro Gln Leu Leu His Ile Ile Pro Cys Pro Arg His Arg Ile 290 295 300 Pro Arg Leu Asn Ile Lys Thr Gly Lys Leu Glu Met Ser Tyr Ser Lys 305 310 315 320 Phe Lys Thr Lys Ser Leu Ser Phe Leu Gly Thr Phe Ile Leu Lys Val 325 330 335 Ala Glu Ser Leu Gln Leu Val Thr Val His Gln Ser Glu Thr Glu Asp 340 345 350 Gly Glu Phe Thr Glu Cys Asn Asn Met Thr Leu Ile Asn Leu Leu Leu 355 360 365 Lys Val Leu Gly Pro Ile His Glu Arg Asn Leu Thr Leu Leu Leu Leu 370 375 380 Leu Leu Gln Ile Leu Gly Ser Ala Ile Thr Phe Ser Ile Arg Tyr Gln 385 390 395 400 Leo Val Arg Leo Phe Tyr Asp Val 405 <210> 5 <211> 408 <212> PRT <213> Rhesus monkey <400> 5 Met Trp Ala Phe Ser Glu Leu Pro Met Pro Leu Leu Val Asn Leu Ile 1 5 10 15 Val Ser Leu Leu Gly Phe Val Ala Thr Val Thr Leu Ile Pro Ala Phe 20 25 30 Arg Gly His Phe Ile Ala Ala Arg Leu Cys Gly Gln Asp Leu Asn Lys 35 40 45 Thr Ser Arg Gln Gln Ile Pro Glu Ser Gln Gly Val Ile Ser Gly Ala 50 55 60 Val Phe Leu Ile Ile Leu Phe Cys Phe Ile Pro Phe Pro Phe Leu Asn 65 70 75 80 Cys Phe Val Lys Glu Gln Cys Lys Ala Phe Pro His His Glu Phe Val 85 90 95 Ala Leu Ile Gly Ala Leu Leu Ala Ile Cys Cys Met Ile Phe Leu Gly 100 105 110 Phe Ala Asp Asp Val Leu Asn Leu Arg Trp Arg His Lys Leu Leu Leu 115 120 125 Pro Thr Ala Ala Ser Leu Pro Leu Leu Met Val Tyr Phe Thr Asn Phe 130 135 140 Gly Asn Thr Thr Ile Val Val Pro Lys Pro Phe Arg Pro Ile Leu Gly 145 150 155 160 Leu His Leu Asp Leu Gly Ile Leu Tyr Tyr Val Tyr Met Gly Leu Leu 165 170 175 Ala Val Phe Cys Thr Asn Ala Ile Asn Ile Leu Ala Gly Ile Asn Gly 180 185 190 Leu Glu Ala Gly Gln Ser Leu Val Ile Ser Ala Ser Ile Ile Val Phe 195 200 205 Asn Leu Val Glu Leu Glu Gly Asp Cys Arg Asp Asp His Val Phe Ser 210 215 220 Leu Tyr Phe Met Ile Pro Phe Phe Phe Thr Thr Leu Gly Leu Leu Tyr 225 230 235 240 His Asn Trp Tyr Pro Ser Arg Val Phe Val Gly Asp Thr Phe Cys Tyr 245 250 255 Phe Ala Gly Met Thr Phe Ala Val Val Gly Ile Leu Gly His Phe Ser 260 265 270 Lys Thr Met Leu Leu Phe Phe Met Pro Gln Val Phe Asn Phe Leu Tyr 275 280 285 Ser Leu Pro Gln Leu Leu His Ile Ile Pro Cys Pro Arg His Arg Ile 290 295 300 Pro Arg Leu Asn Ile Lys Thr Gly Lys Leu Glu Met Ser Tyr Ser Lys 305 310 315 320 Phe Lys Thr Lys Ser Leu Ser Phe Leu Gly Thr Phe Ile Leu Lys Val 325 330 335 Ala Glu Ser Leu Arg Leu Val Thr Ile His Gln Ser Asp Thr Glu Asp 340 345 350 Gly Glu Phe Thr Glu Cys Asn Asn Met Thr Leu Ile Asn Leu Leu Leu 355 360 365 Lys Ile Phe Gly Pro Ile His Glu Arg Asn Leu Thr Leu Leu Leu Leu 370 375 380 Leu Leu Gln Ile Leu Gly Ser Ala Phe Thr Phe Ser Ile Arg Tyr Gln 385 390 395 400 Leu Val Arg Leu Phe Tyr Asp Val 405 <210> 6 <211> 408 <212> PRT <213> Chimpanzee <400> 6 Met Trp Ala Phe Ser Glu Leu Pro Met Pro Leu Leu Ile Asn Leu Ile 1 5 10 15 Val Ser Leu Leu Gly Phe Val Ala Thr Val Thr Leu Ile Pro Ala Phe 20 25 30 Arg Gly His Phe Ile Ala Ala Arg Leu Cys Gly Gln Asp Leu Asn Lys 35 40 45 Thr Ser Arg Gln Gln Ile Pro Glu Ser Gln Gly Val Ile Ser Gly Ala 50 55 60 Val Phe Leu Ile Ile Leu Phe Cys Phe Ile Pro Phe Pro Phe Leu Asn 65 70 75 80 Cys Phe Val Lys Glu Gln Cys Lys Ala Phe Pro His His Glu Phe Val 85 90 95 Ala Leu Ile Gly Ala Leu Leu Ala Ile Cys Cys Met Ile Phe Leu Gly 100 105 110 Phe Ala Asp Asp Val Leu Asn Leu Arg Trp Arg His Lys Leu Leu Leu 115 120 125 Pro Thr Ala Ala Ser Leu Pro Leu Leu Met Val Tyr Phe Thr Asn Phe 130 135 140 Gly Asn Thr Thr Ile Val Val Pro Lys Pro Phe Arg Pro Ile Leu Gly 145 150 155 160 Leu His Leu Asp Leu Gly Ile Leu Tyr Tyr Val Tyr Met Gly Leu Leu 165 170 175 Ala Val Phe Cys Thr Asn Ala Ile Asn Ile Leu Ala Gly Ile Asn Gly 180 185 190 Leu Glu Ala Gly Gln Ser Leu Val Ile Ser Ala Ser Ile Ile Val Phe 195 200 205 Asn Leu Val Glu Leu Glu Gly Asp Cys Arg Asp Asp His Val Phe Ser 210 215 220 Leu Tyr Phe Met Ile Pro Phe Phe Phe Thr Thr Leu Gly Leu Leu Tyr 225 230 235 240 His Asn Trp Tyr Pro Ser Arg Val Phe Val Gly Asp Thr Phe Cys Tyr 245 250 255 Phe Ala Gly Met Thr Phe Ala Val Val Gly Ile Leu Gly His Phe Ser 260 265 270 Lys Thr Met Leu Leu Phe Phe Met Pro Gln Val Phe Asn Phe Leu Tyr 275 280 285 Ser Leu Pro Gln Leu Leu His Ile Ile Pro Cys Pro Arg His Arg Ile 290 295 300 Pro Arg Leu Asn Ile Lys Thr Gly Lys Leu Glu Met Ser Tyr Ser Lys 305 310 315 320 Phe Lys Thr Lys Ser Leu Ser Phe Leu Gly Thr Phe Ile Leu Lys Val 325 330 335 Ala Glu Ser Leu Gln Leu Val Thr Val His Gln Ser Glu Thr Glu Asp 340 345 350 Gly Glu Phe Thr Glu Cys Asn Asn Met Thr Leu Ile Asn Leu Leu Leu 355 360 365 Lys Ile Leu Gly Pro Ile His Glu Arg Asn Leu Thr Leu Leu Leu Leu 370 375 380 Leu Leu Gln Ile Leu Gly Ser Ala Ile Thr Phe Ser Ile Arg Tyr Gln 385 390 395 400 Leu Val Arg Leu Phe Tyr Asp Val 405 <210> 7 <211> 408 <212> PRT <213> Domestic dog <400> 7 Met Trp Ala Phe Pro Glu Leu Pro Met Pro Leu Leu Val Asn Leu Val 1 5 10 15 Gly Ser Leu Leu Gly Phe Val Ala Thr Val Thr Leu Ile Pro Ala Phe 20 25 30 Arg Gly His Phe Ile Ala Ala His Leu Cys Gly Gln Asp Leu Asn Lys 35 40 45 Thr Gly Arg Gln Gln Ile Pro Glu Ser Gln Gly Val Ile Ser Gly Ala 50 55 60 Val Phe Leu Ile Ile Leu Phe Cys Phe Ile Pro Phe Pro Phe Leu Asn 65 70 75 80 Cys Phe Met Glu Glu Gln Cys Lys Ala Phe Pro His His Glu Phe Val 85 90 95 Ala Leu Ile Gly Ala Leu Leu Ala Ile Cys Cys Met Ile Phe Leu Gly 100 105 110 Phe Ala Asp Asp Val Leu Asn Leu Arg Trp Arg His Lys Leu Leu Leu 115 120 125 Pro Thr Ala Ala Ser Leu Pro Leu Leu Met Val Tyr Phe Thr Asn Phe 130 135 140 Gly Asn Thr Thr Ile Val Val Pro Lys Pro Phe Arg Pro Ile Leu Gly 145 150 155 160 Leu His Leu Asp Leu Gly Ile Leu Tyr Tyr Val Tyr Met Gly Leu Leu 165 170 175 Ala Val Phe Cys Thr Asn Ala Ile Asn Ile Leu Ala Gly Ile Asn Gly 180 185 190 Leu Glu Ala Gly Gln Ser Leu Val Ile Ser Ala Ser Ile Ile Val Phe 195 200 205 Asn Leu Val Glu Leu Glu Gly Asp Tyr Arg Asp Asp His Val Phe Ser 210 215 220 Leu Tyr Phe Met Ile Pro Phe Phe Phe Thr Thr Leu Gly Leu Leu Tyr 225 230 235 240 His Asn Trp Tyr Pro Ser Gln Val Phe Val Gly Asp Thr Phe Cys Tyr 245 250 255 Phe Ala Gly Met Thr Phe Ala Val Val Gly Ile Leu Gly His Phe Ser 260 265 270 Lys Thr Met Leu Leu Phe Phe Met Pro Gln Val Phe Asn Phe Leu Tyr 275 280 285 Ser Leu Pro Gln Leu Leu His Ile Ile Pro Cys Pro Arg His Arg Ile 290 295 300 Pro Arg Leu Asn Thr Lys Thr Gly Lys Leu Glu Met Ser Tyr Ser Lys 305 310 315 320 Phe Lys Thr Lys Ser Leu Ser Phe Leu Gly Asn Phe Ile Leu Lys Val 325 330 335 Ala Ala Ser Leu Gln Leu Val Thr Val His Gln Ser Glu Asn Glu Asp 340 345 350 Gly Ala Phe Thr Glu Cys Asn Asn Met Thr Leu Leu Asn Leu Leu Leu 355 360 365 Lys Val Leu Gly Pro Met His Glu Arg Asn Leu Thr Leu Leu Leu Leu 370 375 380 Leu Leu Gln Ile Leu Gly Ser Ala Val Thr Phe Ser Ile Arg Tyr Gln 385 390 395 400 Leu Val Arg Leu Phe Tyr Asp Val 405 <210> 8 <211> 408 <212> PRT <213> Guinea pig <400> 8 Met Trp Ala Phe Ser Glu Val Pro Ile Pro Leu Leu Val Asn Leu Ile 1 5 10 15 Gly Ser Leu Leu Gly Phe Val Ala Thr Leu Thr Leu Ile Pro Ala Phe 20 25 30 Arg Gly His Phe Ile Ala Ala Arg Leu Cys Gly Gln Asp Leu Asn Lys 35 40 45 Thr Asn Arg Gln Gln Ile Pro Glu Ser Gln Gly Val Ile Ser Gly Ala 50 55 60 Val Phe Leu Ile Ile Leu Phe Cys Phe Ile Pro Phe Pro Phe Leu Asn 65 70 75 80 Cys Phe Val Lys Glu Gln Cys Lys Ala Phe Pro His His Glu Phe Val 85 90 95 Ala Leu Ile Gly Ala Leu Leu Ala Ile Cys Cys Met Ile Phe Leu Gly 100 105 110 Phe Ala Asp Asp Val Leu Asn Leu Arg Trp Arg His Lys Leu Leu Leu 115 120 125 Pro Thr Ala Ala Ser Leu Pro Leu Leu Met Val Tyr Phe Thr Asn Phe 130 135 140 Gly Asn Thr Thr Ile Val Val Pro Lys Pro Phe Arg Pro Val Leu Gly 145 150 155 160 Leu His Leu Asp Leu Gly Ile Leu Tyr Tyr Val Tyr Met Gly Leu Leu 165 170 175 Ala Val Phe Cys Thr Asn Ala Ile Asn Ile Leu Ala Gly Ile Asn Gly 180 185 190 Leu Glu Ala Gly Gln Ser Leu Val Ile Ser Ala Ser Ile Ile Val Phe 195 200 205 Asn Leu Val Glu Leu Gln Gly Asp Tyr Arg Asp Asp His Val Phe Ser 210 215 220 Leu Tyr Phe Met Ile Pro Phe Phe Phe Thr Thr Leu Gly Leu Leu Tyr 225 230 235 240 His Asn Trp Tyr Pro Ser Gln Val Phe Val Gly Asp Thr Phe Cys Tyr 245 250 255 Phe Ala Gly Met Thr Phe Ala Val Val Gly Ile Leu Gly His Phe Ser 260 265 270 Lys Thr Met Leu Leu Phe Phe Met Pro Gln Val Phe Asn Phe Leu Tyr 275 280 285 Ser Leu Pro Gln Leu Leu His Ile Ile Pro Cys Pro Arg His Arg Ile 290 295 300 Pro Arg Leu Asn Thr Lys Thr Gly Lys Leu Glu Met Ser Tyr Ser Lys 305 310 315 320 Phe Lys Thr Asn Ser Leu Ser Phe Leu Gly Thr Phe Ile Leu Lys Val 325 330 335 Ala Glu Arg Leu Gln Leu Val Thr Val His Arg Ser Glu Gly Glu Asp 340 345 350 Gly Ala Phe Thr Glu Cys Asn Asn Met Thr Leu Ile Asn Leu Leu Leu 355 360 365 Lys Ile Phe Gly Pro Ile His Glu Arg Asn Leu Thr Leu Leu Leu Leu 370 375 380 Leu Leu Gln Ile Val Gly Ser Ala Val Thr Phe Ser Ile Arg Tyr Gln 385 390 395 400 Leu Val Arg Leu Phe Tyr Asp Val 405 <210> 9 <211> 410 <212> PRT <213> Rattus norvegicus <400> 9 Met Trp Ala Phe Pro Glu Leu Pro Leu Pro Leu Pro Leu Leu Val Asn 1 5 10 15 Leu Ile Gly Ser Leu Leu Gly Phe Val Ala Thr Val Thr Leu Ile Pro 20 25 30 Ala Phe Arg Ser His Phe Ile Ala Ala Arg Leu Cys Gly Gln Asp Leu 35 40 45 Asn Lys Leu Ser Arg Gln Gln Ile Pro Glu Ser Gln Gly Val Ile Ser 50 55 60 Gly Ala Val Phe Leu Ile Ile Leu Phe Cys Phe Ile Pro Phe Pro Phe 65 70 75 80 Leu Asn Cys Phe Val Glu Glu Gln Cys Lys Ala Phe Pro His His Glu 85 90 95 Phe Val Ala Leu Ile Gly Ala Leu Leu Ala Ile Cys Cys Met Ile Phe 100 105 110 Leu Gly Phe Ala Asp Asp Val Leu Asn Leu Arg Trp Arg His Lys Leu 115 120 125 Leu Leu Pro Thr Ala Ala Ser Leu Pro Leu Leu Met Val Tyr Phe Thr 130 135 140 Asn Phe Gly Asn Thr Thr Ile Val Val Pro Lys Pro Phe Arg Trp Ile 145 150 155 160 Leu Gly Leu His Leu Asp Leu Gly Ile Leu Tyr Tyr Val Tyr Met Gly 165 170 175 Leu Leu Ala Val Phe Cys Thr Asn Ala Ile Asn Ile Leu Ala Gly Ile 180 185 190 Asn Gly Leu Glu Ala Gly Gln Ser Leu Val Ile Ser Ala Ser Ile Ile 195 200 205 Val Phe Asn Leu Val Glu Leu Glu Gly Asp Tyr Arg Asp Asp His Val 210 215 220 Phe Ser Leu Tyr Phe Met Met Pro Phe Phe Phe Thr Thr Leu Gly Leu 225 230 235 240 Leo Tyr His Asn Trp Tyr Pro Ser Gln Val Phe Val Gly Asp Thr Phe 245 250 255 Cys Tyr Phe Ala Gly Met Thr Phe Ala Val Val Gly Ile Leu Gly His 260 265 270 Phe Ser Lys Thr Met Leu Leu Phe Phe Met Pro Gln Val Phe Asn Phe 275 280 285 Leu Tyr Ser Leu Pro Gln Leu Phe Gln Ile Ile Pro Cys Pro Arg His 290 295 300 Arg Met Pro Arg Leu Asn Thr Lys Thr Gly Lys Leu Glu Met Ser Tyr 305 310 315 320 Ser Lys Phe Lys Thr Lys Ser Leu Ser Phe Leu Gly Thr Phe Ile Leu 325 330 335 Lys Val Ala Glu Ser Leu Arg Leu Val Thr Val His Arg Gly Glu Ser 340 345 350 Glu Asp Gly Ala Phe Thr Glu Cys Asn Asn Met Thr Leu Ile Asn Leu 355 360 365 Leu Leu Lys Val Phe Gly Pro Thr His Glu Arg Asn Leu Thr Leu Phe 370 375 380 Leu Leu Leu Leu Gln Val Leu Ser Ser Ala Val Thr Phe Ser Ile Arg 385 390 395 400 Tyr Gln Leu Val Arg Leu Phe Tyr Asp Val 405 410 <210> 10 <211> 410 <212> PRT <213> Mus musculus <400> 10 Met Trp Ala Phe Pro Glu Leu Pro Leu Pro Leu Pro Leu Leu Val Asn 1 5 10 15 Leu Ile Gly Ser Leu Leu Gly Phe Val Ala Thr Val Thr Leu Ile Pro 20 25 30 Ala Phe Arg Ser His Phe Ile Ala Ala Arg Leu Cys Gly Gln Asp Leu 35 40 45 Asn Lys Leu Ser Gln Gln Gln Ile Pro Glu Ser Gln Gly Val Ile Ser 50 55 60 Gly Ala Val Phe Leu Ile Ile Leu Phe Cys Phe Ile Pro Phe Pro Phe 65 70 75 80 Leu Asn Cys Phe Val Glu Glu Gln Cys Lys Ala Phe Pro His His Glu 85 90 95 Phe Val Ala Leu Ile Gly Ala Leu Leu Ala Ile Cys Cys Met Ile Phe 100 105 110 Leu Gly Phe Ala Asp Asp Val Leu Asn Leu Arg Trp Arg His Lys Leu 115 120 125 Leu Leu Pro Thr Ala Ala Ser Leu Pro Leu Leu Met Val Tyr Phe Thr 130 135 140 Asn Phe Gly Asn Thr Thr Ile Val Val Pro Lys Pro Phe Arg Trp Ile 145 150 155 160 Leu Gly Leu His Leu Asp Leu Gly Ile Leu Tyr Tyr Val Tyr Met Gly 165 170 175 Leu Leu Ala Val Phe Cys Thr Asn Ala Ile Asn Ile Leu Ala Gly Ile 180 185 190 Asn Gly Leu Glu Ala Gly Gln Ser Leu Val Ile Ser Ala Ser Ile Ile 195 200 205 Val Phe Asn Leu Val Glu Leu Glu Gly Asp Tyr Arg Asp Asp His Ile 210 215 220 Phe Ser Leu Tyr Phe Met Ile Pro Phe Phe Phe Thr Thr Leu Gly Leu 225 230 235 240 Leo Tyr His Asn Trp Tyr Pro Ser Arg Val Phe Val Gly Asp Thr Phe 245 250 255 Cys Tyr Phe Ala Gly Met Thr Phe Ala Val Val Gly Ile Leu Gly His 260 265 270 Phe Ser Lys Thr Met Leu Leu Phe Phe Met Pro Gln Val Phe Asn Phe 275 280 285 Leu Tyr Ser Leu Pro Gln Leu Phe His Ile Ile Pro Cys Pro Arg His 290 295 300 Arg Met Pro Arg Leu Asn Ala Lys Thr Gly Lys Leu Glu Met Ser Tyr 305 310 315 320 Ser Lys Phe Lys Thr Lys Asn Leu Ser Phe Leu Gly Thr Phe Ile Leu 325 330 335 Lys Val Ala Glu Asn Leu Arg Leu Val Thr Val His Gln Gly Glu Ser 340 345 350 Glu Asp Gly Ala Phe Thr Glu Cys Asn Asn Met Thr Leu Ile Asn Leu 355 360 365 Leu Leu Lys Val Phe Gly Pro Ile His Glu Arg Asn Leu Thr Leu Leu 370 375 380 Leu Leu Leu Leu Gln Val Leu Ser Ser Ala Ala Thr Phe Ser Ile Arg 385 390 395 400 Tyr Gln Leu Val Arg Leu Phe Tyr Asp Val 405 410 <210> 11 <211> 1920 <212> DNA <213> Mus musculus <400> 11 gttgcttcct aagagcttct tgctggtcag gagggagggt caggtcctag cgtcctagct 60 gggttttgtt cccgctggcg ccggaatcct ctgcgggttg ggagccgcac tgccggctgc 120 cgaggccacg ggattgttcc tggcttacca gttagctgag taggcggcgg ggcggcggcc 180 accggagggt caccatgtgg gccttcccgg agttgcccct gccgctgccg ctgctggtga 240 atttgatcgg ctcgctgttg ggattcgtgg ctacagtcac cctcatccct gccttccgta 300 gccactttat cgccgcgcgc ctctgtggcc aggacctcaa caagctcagc cagcagcaga 360 tcccagagtc ccagggagtg atcagcggtg ctgttttcct tatcatcctc ttctgcttca 420 tccctttccc cttcctgaac tgcttcgtgg aggagcagtg taaggcattc ccccaccatg 480 aatttgtggc cctaataggt gccctccttg ccatctgctg catgatcttc ctggggtttg 540 ctgatgatgt cctcaatctc cgctggcgcc acaagctgct gctgcccaca gctgcctcac 600 tacctctcct catggtctac ttcacaaact ttggcaatac aaccatcgtg gtgcccaagc 660 ccttccgctg gattctgggc ctgcatttgg acttggggat cctgtactac gtctacatgg 720 ggctgcttgc agtgttctgt accaatgcca tcaacatcct ggcgggcatt aatggcctag 780 aggccggtca gtcactagtc atctctgctt ctatcattgt cttcaacctg gtggaactgg 840 aaggtgatta tcgagatgat catatctttt ccctttactt catgatacca tttttcttta 900 ccaccttggg actgctttac cacaactggt acccgtcccg cgtgtttgtg ggagacacct 960 tctgttactt tgcgggcatg acttttgccg tggtggggat cttgggacac ttcagcaaga 1020 ccatgctgct cttctttatg ccacaagtat tcaatttcct ctactcactg cctcagctct 1080 tccatatcat cccctgccct cgacaccgga tgcccagact caacgcaaag acaggcaaac 1140 tggaaatgag ctattccaag ttcaagacca agaacctctc tttcctgggc acctttattt 1200 taaaggtagc agagaacctc cggttagtga cagttcacca aggtgagagt gaggacggtg 1260 ccttcactga gtgtaacaac atgaccctca tcaacttgct acttaaagtc tttgggccta 1320 tacatgagag aaacctcacc ctgctcctgc tgctcctgca ggtcctaagc agcgccgcca 1380 ccttctccat tcgttaccaa ctcgtccgac tcttctatga tgtctgagct ccctgacagc 1440 tgccctttac ctcacagtct ccattggacc tcagccagga ccagcctctg tctggtccga 1500 gatgaccctc tggtccaggc ctcgctgaca cttttgttct cagcttctgc catctgtgac 1560 tactgatatc ctggatggac accttgctgg acttgaagtc cgctagttgg actttgccta 1620 gggctttcat cttgccttgc cctccctttc tgtcccatct gcagcctcac caggtgggct 1680 tgtagcctct attatgcaaa tattcgtagc tcagctttca gagcgctaac tctaaaggaa 1740 ttcacctgag ccttgagaga gaacctgggc tagggctaga gttagggcta catactccaa 1800 ggtgacctca catttgacta tcaaatgaag tgttgtgatt gggaagcgta gaggcagggc 1860 catgtgctca gaacggtgac aataaaggac tgccttttac ttgttaaaaa aaaaaaaaaa 1920 <210> 12 <211> 2150 <212> DNA <213> Homo sapiens <400> 12 aagtatccgt tcttggctgc ctttctttaa ttgcgtttcc agtactctct cggtgattct 60 actcttgaac ataggatgaa atttggaatc acacttctct tccacttcca tccccaccct 120 ctaatgccca tattaaaaat ggcggccgcc gccttccgca gtaatggttg ttcagcgaac 180 aagatccggg cggaaacagt agataggcgg gtgcagcggg gcagaacata ggttgcctta 240 gagaggttcc ccggtgtccc gacggcggct caagtcagag ttgctgggtt ttgctcagat 300 tggtgtggga agagcctgcc tgtggggagc ggccactcca tactgctgag gcctcaggac 360 tgctgctcag cttgcccgtt acctgaagag gcggcggagc cgggcccctg accggtcacc 420 atgtgggcct tctcggaatt gcccatgccg ctgctgatca atttgatcgt ctcgctgctg 480 ggatttgtgg ccacagtcac cctcatcccg gccttccggg gccacttcat tgctgcgcgc 540 ctctgtggtc aggacctcaa caaaaccagc cgacagcaga tcccagaatc ccagggagtg 600 atcagcggtg ctgttttcct tatcatcctc ttctgcttca tccctttccc cttcctgaac 660 tgctttgtga aggagcagtg taaggcattc ccccaccatg aatttgtggc cctgataggt 720 gccctccttg ccatctgctg catgatcttc ctgggctttg cggatgatgt actgaatctg 780 cgctggcgcc ataagctgct gctacctaca gctgcctcac tacctctcct catggtctat 840 ttcaccaact ttggcaacac gaccattgtg gtgcccaagc ccttccgccc gatacttggc 900 ctgcatctgg acttgggaat cctgtactat gtctacatgg ggctgctggc agtgttctgt 960 accaatgcca tcaatatcct agcaggaatt aacggcctag aggctggcca gtcactagtc 1020 atttctgctt ccatcattgt cttcaacctg gtagagttgg aaggtgattg tcgggatgat 1080 catgtctttt ccctctactt catgataccc ttttttttca ccactttggg attgctctac 1140 cacaactggt acccatcacg ggtgtttgtg ggagatacct tctgttactt tgctggcatg 1200 acctttgccg tggtgggcat cttgggacac ttcagcaaga ccatgctact attcttcatg 1260 ccccaggtgt tcaacttcct ctactcactg cctcagctcc tgcatatcat cccctgccct 1320 cgccaccgca tacccagact caatatcaag acaggcaaac tggagatgag ctattccaag 1380 ttcaagacca agagcctctc tttcttgggc acctttattt taaaggtggc agagagcctc 1440 1500 atgaccctca tcaacttgct acttaaagtc cttgggccca tacatgagag aaacctcaca 1560 ttgctcctgc tgctgctgca gatcctgggc agtgccatca ccttctccat tcgatatcag 1620 ctcgttcgac tcttctatga tgtctgagtc ccttgatcat tgtcctttac ctcacagtct 1680 ctaggattcc tgactcaggc tgacctctct ctctggtccc agactgcctc cttgcccagg 1740 cctctctcac tcttcatact cctccagatt ttgttctcag cattttcctt tctctgtgat 1800 cattggcatc ctgggcgttt cttgccctct gctgactact gattggattt tacctatggc 1860 tttctgcaac ttgctactct ctccctctcc atcccatctt tgcagcctca tagggtggga 1920 tacagcagct ttttttgcag ttatccacac tcacatttca gagtcctgac tctcaaggaa 1980 ccactggttt ttgggataga acttgggcca gggctaggaa cacaggctcc acggtgacat 2040 gtcatttgat tgtaaattaa gtgttctgat tagtaagaac tagcagggg gccacatgct 2100 ctcaatggag acaataaagt gttgtctttt tcttattgtt taaaaaaaaa 2150 <210> 13 <211> 1840 <212> DNA <213> Rattus norvegicus <400> 13 gggggctggc gccggaatcc tctgagtgta gggagctgca ctgctggctg ccgaggcctc 60 tggtttgttc ctggcttacc aagttagctg agtaggcggc ggagcggcgg cccccggagg 120 gtcactatgt gggccttccc ggagttacct ctgccgctgc cgctgttggt gaatttgatc 180 ggatcgctgt tgggatttgt ggctaccgtc accctcatcc ctgccttccg tagccacttt 240 atcgccgcgc gtctctgtgg ccaggacctc aacaagctca gccggcagca gatcccagag 300 tcccagggag tgatcagcgg tgctgttttc cttatcatcc tcttctgctt catccctttc 360 cctttcctga actgctttgt ggaggagcag tgtaaggcat tcccccacca tgaatttgtg 420 gccctgatag gtgccctcct tgctatctgc tgcatgatct tcctgggttt tgctgatgac 480 gtactcaatc tgcgatggcg tcataagctg ctgctcccca cagctgcctc actacctctc 540 ctcatggtct acttcactaa ctttggcaat acaaccattg tggtgccgaa gcccttccgc 600 tggattcttg gcctgcattt ggatttgggc atcctgtact atgtctacat gggactgctt 660 gcagtgttct gtaccaatgc catcaacatc ctagcgggaa ttaatggcct agaggctggt 720 caatcactag tcatctctgc ttctattatt gtcttcaacc tggtggagct ggaaggtgat 780 tatcgggacg atcatgtctt ttccctctac ttcatgatgc catttttttt taccaccttg 840 ggattgctgt accataactg gtacccgtct caggtgtttg tgggagacac cttctgttat 900 tttgctggca tgacctttgc cgtggtggga atcttgggac acttcagcaa gaccatgctg 960 ctcttcttta tgccacaagt attcaatttc ctctactcac tgcctcagct ctttcagatc 1020 atcccctgcc ctcgacaccg tatgcccaga ctcaatacga agacaggcaa actggagatg 1080 agctattcca agttcaagac caagagcctc tctttcttgg gcacgtttat tttaaaggta 1140 gcagagagcc tccggctggt gacagttcac cgaggggaga gtgaggatgg tgccttcact 1200 gagtgtaaca acatgaccct catcaacttg ctacttaaag tctttgggcc tacacatgag 1260 agaaacctca cactgttcct gctgctcctg caggttctga gcagcgctgt caccttctcc 1320 attcgttacc agctcgtccg actcttctat gatgtctgag ctccctgacg actgcccttt 1380 accacacagt ctccattgga cctcagccag gacccacctc tgtccgctcc gaccgccttc 1440 tggtccaggc tcagcttctg ccgtcatctg tgactactga catcctggat ggactcctta 1500 gtggacttga cgtccactag ttggacttgc ctatgctttc ttgagtttgc tactccctcc 1560 ctttctgcag cctcaccagg tgggcctgta gcatctttta tgcaaatatt catggctcaa 1620 ctttcagaac cctaactcta aaggaatccc ctgggccttg agagagaacc tgggctaggg 1680 ctagagttag ggcaacatac tccaaggtaa cctcacatct gactatcaaa ttaagtgttc 1740 tgattaggaa gagcagaggc agggccatgt gctcagaatg gtgacaataa aggattgcct 1800 tttacttgcc aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1840 <210> 14 <211> 5424 <212> DNA <213> Rhesus monkey <400> 14 tattaaaaat ggcggctgcc gccctccgca gtaatagttg ttcagcgaat aaaatccggg 60 cggaaacagt aggtaggctg gttcagtggg ggcagaacct aggttgcctt agagaggttc 120 ttcgaggtcc cgagggcggc tcaagtcaga gttgttggat tctgctcaga ttggtgtggg 180 aagagcctgc ctgtggggag cggccactcc atactgctga ggcctcagga ctgctgctca 240 gcttgccagt tacctgaaga ggcggcggag ccgggcccct gaccggtcac catgtgggcc 300 ttctcggaat tgcccatgcc gctgctggtc aatttgatcg tctcgctgct gggatttgtg 360 gccacagtca ccctcatccc agccttccgg ggccacttca ttgctgcgcg cctctgtggt 420 caggacctca acaaaaccag ccgacaacag atgtgagcag cggcacacgg ttccgggcag 480 ggggcaaggg ctaaggaagg agtggctagg gcaggggcgg ggaccggggt gtttgaccac 540 acgtgaaaac tcagaactaa cccaggcagc ctggaactcg gagaggtgat gagcagaact 600 tattcgcatt ggggaaagga tgggtaggga accttgggta tatcagggac tctagcagtg 660 gtgctttcct ccctccgccc ccctcaccac ttcccaaaat aaaaaaccag gaatgagaag 720 accgctttgg gttattgtaa cacctgcact agtgagtgac cacaccccct ttcctctttc 780 ccctcgcccc cttgctgctg ggccacagcc cagaatccca gggagtgatc agcggtgctg 840 ttttccttat catcctcttc tgcttcatcc ctttcccctt cctgaactgc tttgtgaagg 900 agcagtgtaa ggcattcccc caccatgaag taagtgggtt cgtgggggtt gttgcctgtg 960 gctgggacct gggaggtacc tgagagaatt ggtgttattt gggcttgtgg ggaggggcta 1020 agaaatgata agaaaagaca agaattctta aaaggtgaaa tgggagcagg cttgagtcat 1080 ggacctgccc tagcctcccc cagtttgtgg ccctgatagg tgccctcctt gccatctgct 1140 gcatgatctt cctgggcttt gcggatgatg tactgaatct gcgctggcgc cataagctgc 1200 tgctacccac agctgcctca ctacctctcc tcatggtcta tttcaccaac tttggcaaca 1260 cgaccattgt ggtgccgaag cccttccgcc cgattcttgg cctgcatctg gacttgggta 1320 ggtagtcctg ccactgctac tcctatggca cctacttcag ggcacccttc ctggtgcttc 1380 acattctcct tcaagtgttc cttttctgtc tctgtgtctt cccagatcct ttctggtagc 1440 ccttcatcct atcctctgtc ctcaccactt ttctaaatcc tcctccccta ggtggcacta 1500 cttttcctac catctctccc ttcaggaatc ctgtactatg tctacatggg gctgctggca 1560 gtgttctgta ccaatgccat caatatccta gcaggaatta atggcctaga ggctggccag 1620 tcactagtaa tttctgcttc catcattgtc ttcaacctgg tagagttgga aggtaggtgg 1680 gattgggggt ggggagagag aagtctgaat gttaaaggtg tggcctgata tatgactttg 1740 ggaaattcag ggaaaaaaag caatatgcgt agtaattata gaagataagg gaggctactt 1800 actttgcaaa taatgcagat ttattgaaag tgagaaagaa aaatagcagc cgtgtcattt 1860 atagctggat tggcactaac agctaggcca tgatcttctc ccattgaata taaacaattt 1920 cacagaacct caacgttaca cagggtcatt ctgtgaccat gatggaggaa gaccaaaact 1980 cgacccctcc ctctataatc ctgtttgagc acagataaaa ccacaaaaac actgagcaac 2040 ccacaaaatg gccaagatcc tctctctctg ttaacgtgag ccatgagcga ctgctgcggc 2100 tttccaataa caactcagtt cctaccacct tttattttgt tttttgagac agggtctccc 2160 tctgtcaccc aggctggaga gcagtggcac gatcttagct cactgcatcc tctgactcaa 2220 acgatcctcc tgccccagac tcccaggtag ctggggctac aggcatgcgc caccacacct 2280 ggcaaatttt tgtattttt gtagagacag ggtttcacca cgttgcctag gctggtcttg 2340 aactcctggg ccgaagtgat ttgtcagcct tggcctccca aagtgctggg attacactct 2400 tgagccactg tggccagcca gttcctacca cttcttagat aaacattaaa atgcttgatc 2460 agagaattat tgttgttttc ttttcttttt cttttttttt tttttttgag acggagtttc 2520 actcttgccc aggctggagt gcaatggtgc gatctcggct cactgcaacc tccacctccc 2580 aggttcaggc gattctcctg cctcagcctc cctagtagct gggattacag gcatgtgcca 2640 tcacgcccag ctagttttgt atttttagta gagatgggat ttctccatgt tggtcaagct 2700 ggtctcaaac tccagacctc aggtgatccg cccacctcgg cctcccaaag tgctgggatt 2760 acaggtgtga gcaccgcac ccggccatga attacacctg ctttctaaca gcacccaatc 2820 cagagcaaaa ctcttacttt cttttaccct ctcccaaaat acccaaaact gcaagcccct 2880 cctaacactc tcttactgag acattccgtg gttcccaatg gtgtgtggtt tctgaagtct 2940 ccctttttac aacaagtcat taaacctagc ttcgaactat agatgtgttt ctagtggtct 3000 ttggctgatg gacatcaaca aatgtttatt aaagctaagt actttttaaa catgatcgta 3060 tttaaatctt gtaatggttt tatgtggcac atgttataat cagccctgtt ttacagatga 3120 gtaaacagat ttagagaagt taaatgtgtc atgatcaaga tcaaggtcac aaagctaaga 3180 agtaaagttg gtgtccaaac tgacatcaga atgggctaaa ccaaatttaa gacagtaact 3240 agtttggaag gctgcatgaa agaggtggaa tattgggaat tgccttgggt gacataaaaag 3300 gggtattgag ttcttgaaag tgacttgggt gaggtggatg atacagctgt aaacagaact 3360 tagacaaaaa taggaccatg gtatgcagaa gaagtgggta ttaattttcc cttctttctt 3420 ccttgcttcc aaaggtgatt gtcgggatga tcatgtcttt tccctctact tcatgatacc 3480 ctttttttc accactttgg gattgctgta ccacaactgg tagtagggcc tatggataag 3540 gggaaaaggg gaaactacc cgaacacatg gcaaagatgg cccttatcat aacccacctt 3600 gtggtggaga ggttaaaacct gtgcatacct ccatggaatt ttctgtgtct tcagttggtc 3660 gtattctgaa atttctccct acccaacagt atttggggat gagtgcgtgg aggtcccagg 3720 aatagatgaa ttcagggcct tggatcctgc agagttgctg cacaactgga gtctcctcta 3780 agtcagaact agggtcaggg ctagtacagt gcccataggg tgtgatgtga gagaaaggat 3840 tggtaatgcc tcttgccact ggctcggatc ctctccccca cacaggtacc catcacgggt 3900 gtttgtggga gacaccttct gttactttgc tggcatgacc tttgccgtgg tgggcatctt 3960 gggacacttc agcaagacca tgctactatt cttcatgccc caggtgttca acttcctcta 4020 ctcactgcct cagctcctgc atatcatccc ctgccctcgc caccgcatac ccaggtagcc 4080 gctttggggc ttgaaatgga catcatagcc ttttcacttg ggatatctaa tgccagcctt 4140 tacattgctg tgcaaaggga gtgggcccaa agaagggcta tttccatgtg agtaaccctt 4200 tataacttcc aaagcacatt tatttgcatc atctgatact cacagtggtt ctgataacag 4260 caagcagcag agccagaaat agatctcagg ttgactccac attcaatgct cttcctattg 4320 attagccaca gggaggaggg ttcaaatagt ggcccagtca catgaagctg tcttcccccc 4380 gcagactcaa tatcaagaca ggcaaactgg agatgagcta ttccaagttc aagaccaaga 4440 gcctctcttt cttgggcacc tttattttaa aggtaacagg gtaacaagga ggtaaggccc 4500 taggctgcca tcctgacctt gaggaatggg gaacctaggc ctacatcaga tccaagggga 4560 acttggaagc attaaataga tccacattcc taaagcatag gtattagctg aggttctctt 4620 cacctctggt ccctccaggt ggcagagagc ctccggctgg tgacaataca ccaaagtgat 4680 actgaggatg gtgaattcac tgaatgtaac aacatgaccc tcatcaactt gctacttaaa 4740 atctttgggc ccatacatga gagaaacctc acattgctcc tgctgctgct gcaggtgagg 4800 atggggattg ggtttatacc tccttgtctc cctttctccg tgattcttat tccagtccat 4860 ttctccttgc agatcctggg cagtgccttc accttctcca ttcgatatca gctcgttcga 4920 ctcttctatg atgtctgagt cccttgatca ttgtccttta cctcacagtc tctaggattc 4980 ctgactcagg ctgacctctc tctggtccca gactgcctcc ttgcccaggc ctctctcact 5040 cttcatactc ttccagattt tgttctcagc attttctttt ccctgtgatc actggcatcc 5100 tgggcgtttc ttgcccccta ctgtctactg attggatttt acttatgact ttctgcaact 5160 tgctactctc cctctccatc ctgtctttgc agcctcacag ggtgggatac agaagttttt 5220 tttttgcagt tatccacagt cacatttcag agtcctgact ctcaaggaac tactggtttt 5280 tgggatagaa cttgggccag ggctagggac acaggctcca cagtgacctg ttatttgatt 5340 gtaaattaag tgttctgatt agtaagaagt aagcaggggg ccacatgctc tcaatggaga 5400 caataaagtg ttgtctattt ctta 5424 <210> 15 <211> 5894 <212> DNA <213> Chimpanzee <220> <221> misc_feature <222> (3408)..(3408) <223> n is a, c, g or t <400> 15 aaaccgtagc tgcgtttccg ggaactgagt tgtgtttacc ttggcttccg actatgttgg 60 caacaggttt cctgcaagaa actggcgcgt ctccacaccc tcgtccctcc tccccacccc 120 ctgcctttca atagccatct tcctggagcc ggaggcatcc cagattaagg gagaggtacg 180 ggccctttaa gcttgaccta tggaggcgga cggagctaaa actgacgtgg aaccggaatg 240 tgagcggtgt cagacacgtg gtacaaggag gcattcatct tggaaccggg caattggcat 300 ttccgctctg ggtagtacat cttaacata atgttaggga agtatccgtt cttggctgcc 360 tttctttaat tgcgtttcca gtactctctc ggtgattcta ctcttgaaca taggatgaaa 420 tttggaatca cacttctctt gcacttccat ccccaccctc taatgcccat attaaaaatg 480 gcggccgccg ccttccgcag taatggttgt tcagcgaaca agatccgggc ggaaacagta 540 gataggcggg tgcagcgggg cagaacatag gttgccttag agaggttctc cggtgtctcg 600 agggcggctc aagttagagt tgttgggttt tgctcagatt ggtgtgggaa gagcctgcct 660 gtggggagcg gccactccat actgctgagg cctcaggact gctgctcagc ttgcccgtta 720 cctgaagagg cggcggagcc gggcccctga ccggtcacca tgtgggcctt ctcggaattg 780 cccatgccgc tgctgatcaa tttgatcgtc tcgctgctgg gatttgtggc cacagtcacc 840 ctcatcccgg ccttccgggg ccacttcatt gctgcgcgcc tctgtggtca ggacctcaac 900 aaaaccagcc gacagcagat gtgagcagcg gcacacgggt ctgggcaggg ggcaagggct 960 aaggaaggag tggctagggc aggggcgggg accggggtgc ttgaccacac gtgaagactc 1020 agaactaacc caggcagcct ggaactcgga gaggtgatga gcagaactta ctcgcattgg 1080 ggaaaggatg ggtagggacc cttgggtata tctgggactc tggcagtggt gctttcctcc 1140 ctccgccccc ctcaccactt accagaataa aaaaccggga atgagaagac cactttgggt 1200 tattgtaaca cctgcactag tgagtgacca cgcccccttt gctcttcccc ctcgccccct 1260 tgctgctggg ccacagccca gaatcccagg gagtgatcag cggtgctgtt ttccttatca 1320 tcccttctg cttcatccct ttccccttcc tgaactgctt tgtgaaggag cagtgtaagg 1380 cattccccca ccatgaagta agtgggttcg tgggggtgat tgcctgtggc tgggacctgg 1440 gaggtacctg agagaattgg ggttatttgg gcttgtgggg aggggctaag aaattatcag 1500 aaaagacagg aattcttaaa aggtggaatg ggagcaggct tgagtcatgg acctgcccga 1560 gcccccccca gtttgtggcc ctgataggtg ccctccttgc catctgctgc atgatctttcc 1620 tgggctttgc ggatgatgta ctgaatctgc gctggcgcca taaactgctg ctacctacag 1680 ctgcctcact acctctcctc atggtctatt tcaccaactt tggcaacacg accattgtgg 1740 tgcccaagcc cttccgcccg atacttggcc tgcatctgga cttgggtagg tagtcctacc 1800 actgctgccc ctatggcacc tacttcaggg aacccttcct ggtgctccac attctcctcc 1860 aagtgttcct ttctgtctc tgtgtcttc cagatccttt ctggtagccc ttcatcctat 1920 cgtccgtcct caccactttt ctaaaaattc ttaaatcctc ctcccctagg tggcactact 1980 tcttttccta ccatttctcc ccgcaggaat cctgtactat gtctacatgg ggctgctggc 2040 agtgttctgt accaatgcca tcaatatcct agcaggaatt aacggcctag aggctggcca 2100 gtcactagtc atttctgctt ccatcattgt cttcaacctg gtagagttgg aaggtaggtg 2160 ggattggggg tggggagaga gaagtctgag cattaaaggt gtggcctgat atatgacttt 2220 gggaaattca gggaaaaaaa gcaatatgtg tagtaattat agaagataag ggaagctact 2280 tactttgcaa ataacaatgc agatttatta aaagtgagaa agaaaatag cagccctgtc 2340 atttatagct ggattggcac tatagctag gccatgatct tctcccattg aatataaaca 2400 gtttcacaga accccaacgt tacacagggt cattctgtga ccatgatgga gcaagactaa 2460 aactagaccc ctccctctgt aatcatgttt gagcacaggc aaaaccacaa gaacactgag 2520 caacccacaa aatggccaag atcccctctc tcggctaaca tgagcgactg ctgctgctct 2580 ccaataacaa ctcagttcct accacttctt tttttttttt ttgagacagg gtctccctct 2640 gtcatgcagg ctggagagca gtggcgcaat cttagctcac tgcatcctct gactcaaacg 2700 atcctcctgc cccagcctcc caagtagctg gggctacagg catgtgccac cacacctggc 2760 aaatttttgt attttttgta gagacagggt ttcaccatgt tccctaggct ggtcttgaac 2820 tcctggactc aagtgatctg ccaggcctcc caaagtgctg ggattcactc ttgagccact 2880 gtgcccagcc agttcctacc atttcttaaa taaacattaa aatgcttgat catagaatta 2940 ctcttgcttt cttttctttt cttttctttt ttttttgaga cggagttttg ttcttgccca 3000 ggccggagta caatggtgcg atctcggctc accgcaacct ccgcctccca ggttcaagcg 3060 attctcctgc ctcagcctcc ctagtagctg ggattacagg cacgtgccac cacgcccagc 3120 taattttgta tttctagtag agacggggtt tctccatgtt ggtcaggctg gtctcgaact 3180 cctgacctca ggtgatctgc ctgcttcagc ctcccaagt gctgggatta caggcgtgag 3240 tcaccgcacc cggccatgaa ttactcctgc ttctacag cacccagtcc agagcaaaac 3300 tactttcttt caccctctcc caaataccc aaaaaaaacg ctactacaag cccctcctaa 3360 caccctctta ctgagacatt ccgtggttcc caatgtgtg tggtttcnga agtctccctt 3420 tttacaacaa gtcattaaac ctagctttga gctatagatg tgtttctgat ggtcttggct 3480 gatgaacatc aacaagtgtt tattaagct aagtacttt taacactt cttatttaa 3540 tcttgtaatg gttttatgtg gcagatgtta taatcagccc tgttttacag atgagaaaac 3600 aggcttagag aagtcaatg tgtcatgatc aagatgaagg tcacaagct aagaagtaaa 3660 gttggtatcc aaacttacat cagaatgggc taaaccaat ttaagatagt aactagtttg 3720 gaaggctgca cgaagaggt ggaatattgg gattgcctt gggtgacata aaggagtat 3780 tgagttctta aaagtgactt gggtgaggtg gatgataca gctgtaaca gaacttagac 3840 aaaatagga cagaggttg cagaggaagt gggtattaac ttttccttct ttctccttg 3900 cttccaaagg tgattgtcgg gatgatcatg tcttttccct ctacttcatg ataccctttt 3960 ttttcaccac tttgggattg ctctaccaca actggtaagt aggcctgtgg ataaggggac 4020 aactacctga acacatggca aagatggccc ttatcataac ccaccttgtg gtggtgaagc 4080 taaacctgcg catacctcta tggagttttc tgcgtcttca gttggtagta ttctgaaatt 4140 tctctctacc cagtagtagt tagggatgag tgcgtggagg ccccaggaat agttgaattc 4200 agggccttgg atcctgcaga gttgctgcac aactggagtc tcctctgagt cagaactagg 4260 gtcagggcta gtccagtgcc catagggtgt gatgtgagag aagggattgg taatgcctct 4320 tgccactggc tcggatcctc ttcccccaca caggtaccca tcacgggtgt ttgtgggaga 4380 taccttctgt tactttgctg gcatgacctt tgccgtggtg ggcatcttgg gacacttcag 4440 caagaccatg ctactattct tcatgcccca ggtgttcaac ttcctctact cactgcctca 4500 gctcctgcat atcatcccct gccctcgcca ccgcataccc aggtagccgc tttggggctt 4560 gaaatggaca tcatagcctt ttcacttggg atatctaatg ccagcctata catttgctgt 4620 gcaaagggag tgggcccaaa gaagggctat ttccatgtga gtagcccttt ataacttaca 4680 aagcacattt atttgcataa tctgctacag tggttctgat aacagtaagc agcagagcca 4740 gaaatagatc tcaggttgac tccacattca atgctcttcc tattagccac agggaggagg 4800 gttcaaatag tggcccagtc acatgaagct atcttccccc cgcagactca atatcaagac 4860 aggcaaactg gagatgagct attccaagtt caagaccaag agcctctctt tcttgggcac 4920 ctttatttta aaggtaacag ggtaacaagg aggtaaggcc ctaggctgcc atcctgacct 4980 tgaggaatgg ggaacctagt cctacatcag atccaagggg aacttgaaag cattaaatag 5040 atccacattc ctaaagcata ggtattagct gaggttctct tcacctctgg tccctccagg 5100 tggcagagag cctccagctg gtgacagtac accagagtga gactgaagat ggtgaattca 5160 ctgaatgtaa caacatgacc ctcatcaact tgctacttaa aatccttggg cccatacatg 5220 agagaaacct cacattgctc ctgctgctgc tgcaggtgag gatgggaatc gagttatac 5280 ctccgtgtct cccttctgc gtgattctta ctccagtcca tttctccttg cagatcctgg 5340 gcagtgccat caccttctcc attcgatatc agctcgttcg actcttctat gatgtctgag 5400 tcccttgatc attgtccttt acctcacagt ctctaggatt cctgactcag gctgacctct 5460 ctctctggtc ccagactgcc tccttgccca ggcctctctc actcttcata ctcctccaga 5520 ttttgttctc agcattttcc tttctctgtg atcattggca tcctgggcgt ttcttgccct 5580 ctactgacta ctgattggat tttacctatg gctttctgcg acttgctact ctctccctct 5640 ccatcccatc tttgcagcct catagggtgg gatacagcag ctttttttgc agttatccac 5700 actcacattt cagagtcctg actctcaagg aaccactggt ttttgggata gaacttgggc 5760 cagggctagg aacacaggct ccacggtgac atgtcatttg attgtaaatt aagtgttctg 5820 attagtaaga actaagcagg gggccacatg ctctcaatgg agacaataaa gtgttgtctt 5880 tttcttattg ttta 5894 <210> 16 <211> 4557 <212> DNA <213> Canis lupus familiaris <400> 16 gtgaggaggc aagtgcggcg ggggacagcc gagggtgcgc gctggaggct cgcgggagtc 60 ctgggggcgc ctcaattcag agttgggttt tgctcaggcc gctgtgggag gatccagcct 120 gtgccgagcg gctgctcctc cccgcggggg gctccgggct accgcccagc tcgcccatta 180 gccgaggcgg cggcagagcg gggcccctgg ctggtcatca tgtgggcctt cccggagttg 240 ccgatgccgc tgctggtgaa tttggtcggc tcgctgctgg gatttgtggc cacggtcacc 300 ctcatccccg ccttccgtgg ccacttcatc gccgcgcacc tctgtggcca ggacctcaac 360 aaaaccggcc ggcagcagat gtgagcggtg gcacccgggt ccggggaggg ggccggcagg 420 gcaagggcgg gacctggggt gcctgacccc gcggacacgc agcgctaacc ccgcagacag 480 ctgcgggctc tgggagacga agggcagcgc tggccaactc tgggaaggga tgttgcagta 540 caggggaccc tcgggtgtat cagggactcc agcgctggtg cccttccacc ccccttcccc 600 gtagatcgct gtaatgcttg ctctagtgag tgaccacgcc ccctctcctc tcccccgccc 660 cctccctttg ctgctgggcc acagcccaga gtcccaggga gtgatcagcg gtgctgtttt 720 ccttatcatc ctcttctgct tcatcccttt ccccttcctg aactgtttta tggaggagca 780 gtgttaaagcc ttcccccatc agaagtaag tgggtgagtt gggggcggtt gcttggggct 840 ggggcctggg agctacctgg gagagttgtg gttattaggg tttgggtgga ggggctgagg 900 aaggagcgaa gagacgggtg tttttgcaag atgatgtggg cataggcttg agcggtgacc 960 tgcccgagcc tcccccagtt cgtggccctg ataggtgcgc tccttgccat ctgctgcatg 1020 attttcctgg gctttgcgga cgatgtactg aatctgcgct ggcgccacaa gctgctgctg 1080 cctacagctg cctcgctacc tcttctttag gtctatttca ccaactttgg caacacgacc 1140 attgtggtgc ccaagccttt ccggccgatt cttggcctgc atctggactt gggtgagtag 1200 ccctgtgact gacgtccctg tggcccttac tttggggcac ccttaccctg ggagataatc 1260 tagcagagca tcattcctgg tgctccagat cctcttccaa gtgtccccat cttgttcctg 1320 tgtcttctca gatccgttct gttggtcctt cgtccaatcc tctgtcctca ccacttttct 1380 cagagata ttcttaagtc ctcatttcta tggatggcac acttcttact ctcttcttcc 1440 cccagggatc ctgtattatg tttacatggg gctgctggca gtgttctgta ccaatgccat 1500 caataccta gcaggaatta atggcctaga ggcaggccag tcgctagtta tttctgcttc 1560 catcatcgtc ttcaatctgg tggagctgga aggtaggtga gagtgggagt ctgagtatta 1620 aggaaactgc ctgatacctg gctttgggga attcaggaa aaaaagc atatattaa 1680 gattaaatgt aaagaaaaac agctctgtca ttgacagctg aattggcact ataggtagg 1740 ccatgtctt ctgctgaaca taaacaattt cacagaactt cacaatcaga cgaggtcact 1800 ctatgtccat gatagagtaa agcaaccca gattcctcca taaacatgtc tgagtatagc 1860 1920 gagcaattgc ttccttctga taacttggtt ctacttagag aaactaaga tgctcataga 1980 attacktcca ctgacagcac ccagcttgg gcaaactt gcctccttcc tttctccccc 2040 aaattactca aaacaatcct atacacatc ttcctaatac ttcctactg aggcatcccc 2100 tggttaccta tggtgcgtgg tctacagtgt ctctcttgtt acacgtcagt aacccagct 2160 ttgactgcag gtgtgttct ggtgtcttt ggctgatgga tatcagtgct tattaaaaca 2220 aaatactctt aaagcattta aactttgtaa tgtggcaagt gttctcatga accatatttt 2280 acagttgagg aaacagaggg cgagagaatt taagtgtgtc atgatcaagg tcacacagtt 2340 agaaagtaaa gctagtattc aaacctgggc tgaatgatct aaaccaaatt gaagacagca 2400 acttgtatta ggaagggttc atgaatgagg tggaatatta ggaattgcct gagtgacaca 2460 aaagaagtag tgagttctgg aatgggactt ggaagaggtg gaaagtacag ctggggacag 2520 aacttgagac agaaatagga cccagttatg cagggggaag taccttatca actcatcctt 2580 ctttcttttt cttcttcccc tgcttccaaa ggtgattatc gggatgatca cgtcttttcc 2640 ctctacttta tgataccctt ttttttcacc accttgggat tgctctacca taactggtaa 2700 gtgggccatg tgaacatgta gcaagtatgg tcctgttggt cctgacccaa ctcctgttgg 2760 agaggctaag cctgcgcaca cctgtattga gtgttttctg gatgcctagt tggtaatatt 2820 cttcaattac tctctaccca gttgcagtta gagacaagtg ctgtggagcc cccaagaaga 2880 gatgaattca gggctttggg ttctggaggc ttgttggaag atctggagtt tcctccgggc 2940 caggactaga gtcagggcta gtccagggtt caggcgtgt aatatgagag aaaagactga tagtgcctcc tgccactggc tcagatcctc tcccccacac aggtacccat cacaggtgtt 3060 tgtggggagat accttctgtt actttgctgg catgacctt gccgtggtgg gcatcttggg 3120 acacttcagc aagaccatgc tactcttctt catgccccag gtgttcaact tcctctactc actgcctcag ctcctgcata tcataccctg ccctcgccac cgcattccca ggtagccact 3240 ttggggctta aaagggacat cttagctttt tcacttgggga tgcataaagc cagccttctg catctgctgt gtaagggga tggggcccaaa ggaggggctct ttccgtgcaa ttagccctta 3420. cccacataat ctgatcagct ctgatcacac agtggtaagc agagccgga acagatcttc aggttgtctg attccacttt cggtactctt cctattaatt 3540. gaccgcagtg tggagagttc ttggagtagt ggcccagtca cataaagctc tcttccccct gcagactcaa taccaagaca ggcaaactgg agatgagcta ttccaagttc aagaccaaga gcctctcttt cttgggcaac tttattctaa aggtaacagg gtaacgaggt aaggctctag gccaccatcc ggaattcagg gcctggggac cctcggcttg catcagatcc aaggggagcc 3720 tggaagcatg aagcagatcc cccattgctg aagcagagtt gaagttctct ccacctctgg 3780 cccctccagg tagcagcgag cctgcagcta gtgacagtgc accagagtga gaatgaggat 3840 ggtgccttca cggagtgtaa caacatgacg ctcctcaact tgctccttaa ggttctcggg 3900 cccatgcatg agaaacct gactctgctc ctgctgctgc tccaggtgtg gtcagggaag 3960 ggctttgctg gctctggtct ccctttctcc atggctctga ctctggtgtg tttctttctc 4020 ctcacagatc cttggcagtg ctgtcacctt ctccatccgg taccagcttg tccggctctt 4080 ctacgatgtc tgagtccccc aatccttgcc cttcactgca tagtctgcag ggttcctgac 4140 tcaggcctgc ctctttctgg gccaggcacg cttccgggcc caggcctctc tcacctctta 4200 cttttctcca gattttgtac ttagcgattc cgttccgctg tgatcgacat cctgggcctg 4260 tcttgccctg tactgactgt tgattggact ttgcctgtgg ctttcttcaa cttgctgctc 4320 tccctctcta tcccatccct gcggcctccc aaagtgggat actgtgcttt ttatgcagtt 4380 atccaccact cggactctcg aggaatatgt tgggcctggg gatagaaccc tggctgggga 4440 gagggacaca ggctcgaaga tcacttgatt atttgaccat aaattaagta ttctgattcg 4500 taagagcaga ttggggggcc aggtgctccc agtggtgaca ataaagtgtt gtctttt 4557 <210> 17 <211> 5374 <212> DNA <213> Chinese hamster <400> 17 caaggcagag cctaggttgc tttataaaac ctcttgggga agcccgaggg cggttcaaat 60 taagagttgt tgggttttgc cccgcctcgc atgtgaggag cggacactgc tcacggctga 120 gacctcgggg ctgcttccca ccagttagct gagaaggctg cggagctgga acctctggcc 180 actcgccatg tgggccttct ctgaggtacc gattccgctg ctggtgaatt tgatcggctc 240 gctgctggga tttgtggcca cgctcaccct catcccggcc tttcgtggcc actttatcgc 300 tgcgcgcctc tgtggccagg acctcaacaa aaccaaccgg cagcagatgt gagcagtggc 360 acacgggtgt cccgggcagg ggccaggggt gggcaaggca caggcgagct ctgaggtgct 420 taaatgtgcg tacgaaccaa atctaactgg agttgtccgg gaccctggga ctcgatggcc 480 agaagtggtt agcactgggg aatgctaagg aaggggaccc ttgagtgaga acatccagcg 540 gcgcctgcct cccccgccc cccactgccc tccgctcca ctgctccccc gcctcactcc 600 tgggaagatc ttttgggtca catggtttt gcactaacca cgcccatttc ttcttccttc 660 tccaccccct tgctgcgggg ccacagccca gaatcccagg gagtgatcag cggtgccgtt 720 ttccttatca tcctgttctg cttcatcccc ttccccttct tgaactgctt tgtgaaggag 780 cagtgtaagg cttccccca ccatgaagta agtgggttcg tgggggcggt tgcctggggc 840 ctgggaggtt cccgagagag ttggggttgt gtggatttga ggaggagggga ctgaggacct 900 agtggaaaag acagaaattt ttgaaagctt gaatggcagt aggcttgagt catgacctgc 960 ccgagcctcc cccagtttgt ggccctcata ggtgcccttc ttgccatctg ctgcatgatt 1020 ttcttgggct tcgcggacga tgtcctgaat ctacgctggc gccataagct gctgctgccc 1080 acagctgcat cactacctct tcttatggtc tattttacca actttggcaa cacaaccatt 1140 gtggtaccca agcccttccg cccagttctt ggcttgcatc tggatttggg tgagtatccc 1200 tgctgctaca gcccctgtgg cacttattc aagtcaccct ccccccaaag gtgcccagca 1260 gagcaccctt cttgatgttc cacactcccc tgtttttgtt ccgtccctgt gaatgctcag 1320 gttctctctt gtgccctgtc attgtgtgtt ctgttttcag aataccgtta gatccttcc 1380 tagctgtcac tgctttttat actatgtctt gcagggatcc tgtactatgt ctacatgggg 1440 ctcctggcag tgttctgtac caatgccatc aatatcctag caggatta cggcctag gccggccagt cattggtcat ctctgcttcc atcattgtct tcaacctggt ggagctgcaa 1560 ggttggtggg aagagaga tctcagtgtt cagagaattg cctgatatat agctttgaga aaaggggggc ttatagaaga tagggaaagc tatttacttt gcaaataaca atgaagagtt acttgagtag gaggaagaaa aatagcagtc tgtcatttat agctggattg gcatgagtag ctagaccatg actgtttcct attggacata aatagtttca tagaacccca gcatgagaga ggggcgccct gaccgtggtg aaacaagaca gaaaccagac ttctcccact gtaatcatgt 1860. ctgaacaccg acaaaagcac aggaacaaag tcagcccaaa catcccttct tgtctaatgt gagagggtat agctctttg cagtaacaac tcagttcctg ctactctta agttgttcag 1980 tcagaaaatt acttctgctt tctgacatca ggcagtccag agcacaactt ttccttgcag 2040 cctccccaga accacttaa gtgaatccta tgtaagtcc cttctaacaa cctttagagt 2100 acctgcccag catgaggcct tgggtacagt ccccagtatc tctgtttgca tgcatgtaca 2160 catacccaca tgcacacact gaacttactt attgaaggta agcaattt atttgcattt 2220 tttgtgtgtg tgacaaatt tcactatgta attcagaata gccttgaatt cactatgtag 2280 cctaggccgt cctcgaactt acagtgataa tcctgcctca gcttcctaag tgctaagatt 2340 caagtagc actaccaggc cagctaagaa agcaattttt aaactaggta tggtggcaca 2400 catcactaat tctacactc tgggactt gggcaggag atcatgagtt tggactcagc 2460 ctgggcactt ggtaagtctc tgtttctga aaaaacat ggagtggtga tacacacctg 2520 taatcccagc attcatgagg ctggggcagg aggatcacca caggtcaag acctgcctgg 2580 gttacataag caagttcaag gccagcgtga actacgtagt gagaccctgc ctcaacaaa 2640 caaataaata aataaacatg atcctgagtt tggttcccag tactcccccc aataaatgaa 2700 atgaaatgaa agagctgggg aggcagctta gtgctaaggt ccaggacccc atgtgaaggc 2760 agctgcgtgt atgtgttatc agccctgttt catacactag ataataaaac tggttttcaa 2820 acttaagtca gcatgtctgg acaaagtgaa gactttaact tgtttttgac ggtttcatga 2880 cagtagtgag ctattgggaa ctgcctgggt accatcaaag gaataatgag gggctgggga 2940 tttagctcag cggcataagc gcctgccttg caagcaggca gtcatgagtt tgatccccgg 3000 taccgataaa aaggaaaaag acaaaaaaaa aaggaataat gagtttttga aggtggctcg 3060 ggtgagggga ggtggcaaca gagacaggga tgcgacagac aaaatgaaga gcaggggaca 3120 taggggagat gggtgttcac ttttccttct ttgtcctttg tttcccaagg tgattaccgg 3180 gatgatcatg tcttttccct ctacttcatg ataccgtttt tttcaccac cttgggattg 3240 ctgtatcata actggtaagg aggctgtggc tcagggaaaa ggaaaacaac taactggtca 3300 ttggacaaag atggtcctga tcttaaccca gctcctgaaa gacaggctga acttgcgcat 3360 acttttgctc agtgttttct gggtattcag ttggtggatt gcctcccccc gccccgtttt 3420 ttttttgaga cacctgtggc ctctcgagtg ctaggccagt gctttactac tgagtcttgc 3480 cctctagtat tctcaggttt gttcttttct cagcagttgg agacaagtgc tatggagccc 3540 catch tatggggact tgcgttctgc agacttgcta gaccctcctg tccgaactg gatcagggga gcatgtgtgg tggctcacac ctgtaatccc agcactcagg agactgaggc 3660 aggaggatta ccatgagatc gagggcaccc tcagctcaca tagtgacttt gaggccagcc tggactacat agcgagactc ttgtctccaa aagaaaaaaa aagaaagaat aaaagaacag gggtctgagt tcgtccagta cctagcctgt gtgatgtgag agaaaagact gtgatgcctc 3840 ttggcactgg cttggatcct ttcccccaca caggtaccca tctcaggtgt ttgtgggaga 3900 caccttctgt tactttgctg gcatgacctt tgccgtggta ggcatcttgg gacacttcag 3960 caagaccatg ctgctcttct tcatgccaca ggtgttcaac ttcctctact cgctgcctca gctcctgcat atcatcccct gtcctcgcca ccgtataccc aggtagctgt ttgggggctg 4080 gaaagccttt ctactgggat gtctaacacc aggctctaca tttgctgtgc aaagaatgtg 4140 ggcccatagg aaggctaact tttttcatgt aggtggccct ttaagtttac acagcacgtt 4200 tacttccata atctcattta atactcacag tagttctgat catagagtag taagcagcag 4260 agccagaaat agatctcact ccatgatcag tgtttttctt agtcattaac ggaagaaagt 4320 tttttgagta gtgacccagt cacacgaagc tgtctttccc ctacagactc aataccaaga 4380 caggcaaact ggagatgagc tattccaagt tcaagaccaa cagcctttct ttcttgggca 4440 cctttatttt aaaggtaaca aggtaacgag gaggtaaggc cccaggccac catcctgaac 4500 ttgggacatg ggggacccag gcctacatta gatctagagg gagcttggaa gcattaagca 4560 gagccctgtt cctgacatac aggtattggc tgaagttttt ctgtctgtct ctggtctctc 4620 taggtagcag agagactcca gctagtgaca gtgcaccgga gtgagggtga ggacggggcc 4680 ttcactgagt gtaacaacat gaccctcatc aacttgctgc ttaaaatctt tgggcccata 4740 catgagagga acctcacatt gctcttgctg ctgctacagg tgagcctggg gtgagtttgt 4800 gcctcctcat gtccttttct ctatggttct tattctagtc catttctcct tgcagatcgt 4860 gggcagtgct gtcaccttct ccattcgata ccagcttgtc cgactcttct atgacgtttg 4920 agttcctgaa gattgccctc tgccacactg tctccagggg tcctgctcag gccagccagt 4980 ctggttctgt gggcctctcc caatcttcag tctccttcag atttattccc agcatttttc 5040 ataacctatg attatcaaca tcctgagcca ttttgccct ccagcaacta ctaactggac 5100 tttgcctatg gcctccttca acttgccact ctccctaccc atcacagcca gaggcttgat 5160 gtagcagctt tttgcagat atccacaact cagctttcag agtcctcact ctcaaagaac 5220 atgctgggcc ttgagataga acctgagcta gggctaggga cactggtgca agggtgattt 5280 gatatttgat tataaattaa gtgttctgat tagtaagaca gaaggggagc ctggtgctcc 5340 caacggtgac aataaagtgt taccttttc ttgt 5374

Claims

1. A method for using tunicamycin (Tn) as a selection marker in mammalian cell culture, the method comprising: (a) providing a population of mammalian host cells; (b) introducing a nucleic acid into the cell population of step (a) by transfection, wherein the nucleic acid comprises: (i) a mammalian tunicamycin (Tn) resistance gene, wherein the mammalian tunicamycin (Tn) resistance gene encodes a protein consisting of an amino acid sequence selected from SEQ ID NO: 3 or SEQ ID NO: 4, and (ii) a first gene of interest encoding a first protein of interest; and (c) culturing the cell population of step (b) in the presence of Tn, thereby obtaining a cell population containing cell transfectants containing the nucleic acid.

2. The method of claim 1, wherein the mammalian tunicamycin (Tn) resistance gene comprises a nucleotide sequence selected from SEQ ID NO: 2 or SEQ ID NO:

12.

3. The method according to claim 1, further comprising selecting cell transfectants from the cell population obtained in step (c).

4. The method of claim 1, further comprising culturing the resulting cell population from step (c), expressing the first protein of interest from the first gene of interest, and isolating the first protein of interest from the cell culture.

5. The method of claim 3, further comprising culturing the selected cell transfectants, expressing the first protein of interest from the first gene of interest, and isolating the first protein of interest from the cell culture.

6. The method of claim 1, wherein the nucleic acid is integrated at a target locus within the genome of the host cell population.

7. The method of claim 6, wherein the integration at the target locus is mediated by a recombinase that recognizes a specific site within the target locus, or the integration is mediated by homologous recombination.

8. The method of claim 7, wherein the homologous recombination is promoted by a nuclease that recognizes and cleaves a DNA sequence within the target locus.

9. The method of claim 8, wherein the nuclease is a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or an RNA-guided endonuclease.

10. The method of any one of claims 1 to 9, wherein Tn is present at a concentration of 1 µg / mL, 2.5 µg / mL or 5 µg / mL.

11. The method according to any one of claims 1 to 9, wherein culturing the cell population in step (c) comprises culturing in the presence of continuously increasing concentrations of Tn.

12. The method of claim 11, wherein in step (c), the culturing in the presence of continuously increasing concentrations of Tn comprises culturing at a first Tn concentration of 1 µg / mL, followed by culturing at a second Tn concentration of 2.5 µg / mL or 5 µg / mL.

13. The method according to any one of claims 1 to 9, wherein the method is further characterized by one or more of the following: (i) The first gene of interest is linked to the promoter; (ii) the first gene of interest encodes a glycoprotein; (iii) the mammalian host cell is selected from the group consisting of CHO, COS-7, HEK293, tumor cells, lymphocytes and retinal cells; and (iv) the nucleic acid further comprises a second gene of interest.

14. The method of claim 13, wherein the mammalian host cell is a CHO cell.

15. The method of claim 13, wherein the second gene of interest encodes a glycoprotein.

16. The method of claim 15, wherein the glycoprotein encoded by the second gene of interest is selected from an antibody light chain or an antigen-binding fragment thereof, an antibody heavy chain or an antigen-binding fragment thereof, an Fc fusion protein or a fragment thereof, a ligand, and a receptor or a ligand-binding fragment thereof.

17. A method for preparing a recombinant protein of interest, wherein the method comprises: (a) providing a mammalian host cell comprising an exogenous nucleic acid, wherein the exogenous nucleic acid comprises: (i) a mammalian tunicamycin (Tn) resistance gene, wherein the mammalian tunicamycin (Tn) resistance gene encodes a protein consisting of an amino acid sequence selected from SEQ ID NO: 3 or SEQ ID NO: 4, and (ii) a first gene of interest encoding a first protein of interest; (b) culturing the cell in the presence of Tn to express the first protein of interest.

18. The method of claim 17, wherein the mammalian tunicamycin (Tn) resistance gene comprises a nucleotide sequence selected from SEQ ID NO: 2 or SEQ ID NO:

12.

19. The method of claim 17, wherein Tn is present at a concentration of 1 µg / mL, 2.5 µg / mL, or 5 µg / mL.

20. The method of claim 17, wherein step (b) comprises culturing in the presence of continuously increasing concentrations of Tn.

21. The method of claim 20, wherein in step (b), culturing in the presence of serially increasing concentrations of Tn comprises culturing at a first Tn concentration of 1 µg / mL, followed by culturing at a second Tn concentration of 2.5 µg / mL or 5 µg / mL.

22. The method of claim 17, further comprising isolating a protein of interest expressed by the gene of interest.

23. The method according to any one of claims 17 to 22, wherein the method is further characterized by one or more of the following: (i) The first gene of interest is linked to the promoter; (ii) the first gene of interest encodes a glycoprotein; (iii) the mammalian host cell is selected from the group consisting of CHO, COS-7, HEK293, tumor cells, lymphocytes and retinal cells; and (iv) the nucleic acid further comprises a second gene of interest.

24. The method of claim 23, wherein the mammalian host cell is a CHO cell.

25. The method of claim 23, wherein the second gene of interest encodes a glycoprotein.

26. The method of claim 25, wherein the glycoprotein encoded by the second gene of interest is selected from an antibody light chain or an antigen-binding fragment thereof, an antibody heavy chain or an antigen-binding fragment thereof, an Fc fusion protein or a fragment thereof, a ligand, and a receptor or a ligand-binding fragment thereof.

27. An isolated cell comprising an exogenous nucleic acid, wherein the exogenous nucleic acid comprises: (a) a mammalian tunicamycin (Tn) resistance gene, wherein the mammalian tunicamycin (Tn) resistance gene encodes a protein consisting of an amino acid sequence selected from SEQ ID NO: 3 or SEQ ID NO: 4; and (b) The first gene of interest.

28. The isolated cell of claim 27, wherein the isolated cell is further characterized by one or more of the following: (i) the Tn resistance gene has a nucleic acid sequence selected from the group consisting of SEQ ID NO: 2 or SEQ ID NO: 12; (ii) a first gene of interest is linked to a promoter; and (iii) the cell further comprises an exogenously added second gene of interest.

29. The isolated cell of claim 28, wherein the second gene of interest encodes a glycoprotein.

30. The isolated cell of claim 29, wherein the glycoprotein encoded by the exogenously added second gene of interest is selected from an antibody light chain or an antigen-binding fragment thereof, an antibody heavy chain or an antigen-binding fragment thereof, an Fc fusion protein or a fragment thereof, a ligand, and a receptor or a ligand-binding fragment thereof.

31. A vector comprising a nucleic acid, wherein the nucleic acid comprises: (a) a mammalian tunicamycin (Tn) resistance gene, wherein the mammalian tunicamycin (Tn) resistance gene encodes a protein consisting of an amino acid sequence selected from SEQ ID NO: 3 or SEQ ID NO: 4; and (b) The first gene of interest.

32. The vector of claim 31, wherein the vector is further characterized by one or more of the following: (i) the Tn resistance gene has a nucleic acid sequence selected from the group consisting of SEQ ID NO: 2 or SEQ ID NO: 12; (ii) a first gene of interest is linked to a promoter; and (iii) the vector further comprises an exogenously added second gene of interest.

33. The vector of claim 32, wherein the second gene of interest encodes a glycoprotein.

34. The vector of claim 33, wherein the glycoprotein encoded by the exogenously added second gene of interest is selected from an antibody light chain or an antigen-binding fragment thereof, an antibody heavy chain or an antigen-binding fragment thereof, an Fc fusion protein or a fragment thereof, a ligand, and a receptor or a ligand-binding fragment thereof.

35. The isolated cell according to any one of claims 27 to 30, wherein: (i) the exogenous nucleic acid is integrated into a target locus within the genome of the cell; and / or (ii) The cells are selected from CHO, COS-7, HEK293, tumor cells, lymphocytes and retinal cells.

36. The isolated cell of claim 35, wherein the cell is a CHO-K1 cell.

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