T4 DNA ligase variants with increased ligation efficiency

Abstract

The present invention relates to the field of T4 DNA ligase variants, and in particular to T4 DNA ligase variants with increased ligation efficiency, including: a mutant T4 DNA ligase or a biologically active fragment thereof, which has greater activity than wild-type T4 DNA ligase. The mutant T4 DNA ligase or biologically active fragment has one or more substitutions different from the wild-type as more fully described in the Summary of the Invention with reference to the sequence table.

Description

Technical Field

[0001] The present application relates to the field of T4 DNA ligase variants, and in particular, to a mutant T4 DNA ligase or a biologically active fragment thereof. Background Art

[0002] Ligase is commonly used in molecular biology to form phosphodiester bonds between double-stranded nucleic acid fragments at the intersection of juxtaposed 5' phosphate and 3' hydroxyl termini. By designing complementary overhangs between each double-stranded fragment, the ligation can be made both position-specific and directional. This allows the specific integration of DNA or RNA fragments into larger vectors, which is required for most molecular biology studies.

[0003] Among the commercially available ligases, T4 DNA ligase is a versatile enzyme that catalyzes the bonds that join double-stranded DNA or RNA at sticky and blunt ends, has a fast ligation speed, and also repairs mismatches present in nicked DNA by ligation. It can also join sticky or blunt ends of DNA, RNA, and RNA-DNA hybrids (but not single-stranded nucleic acids). Compared with E. coli DNA ligase, it can also join blunt-ended DNA more efficiently.

[0004] Because T4 DNA ligase is fundamental to many molecular biology protocols and is in constant demand, it accounts for a large share of revenue for many life science product companies due to its importance in the molecular biology reagents market. Increasing the activity of T4 DNA ligase benefits the manufacturing of T4 DNA ligase itself, thereby reducing the number of production runs required to produce large quantities of the final product. Increasing the activity of T4 DNA ligase also increases the usefulness of the enzyme by either reducing the amount of enzyme required or reducing the overall time required for complete ligation of substrates, depending on the goals of the user. Summary of the invention

[0005] The present invention relates to engineered T4 DNA ligase mutants that exhibit enhanced ligation activity compared to wild-type T4 DNA ligase. The following T4 DNA ligase mutants (having substitutions at the specified positions, and wherein the amino acid sequence of each mutant is in the sequence identification number after the specified substitution, in addition to the sequence in the sequence listing, each mutant also includes a tag sequence at its C-terminus: GLGSGSSGHHHHHH (SEQ ID NO:32); and wherein the DNA sequence of each such mutant is also shown as the odd-numbered sequence identification number in the sequence listing preceding the amino acid sequence identification number of each mutant) were identified as having such enhanced ligation activity: E23K (SEQ ID NO:4), E88K (SEQ ID NO:6), K98E (SEQ ID NO:8), E132K (SEQ ID NO:10), E143K (SEQ ID NO:12), E173K (SEQ ID NO:14), E240K (SEQ ID NO:16), E271K (SEQ ID NO:18), K306E (SEQ ID NO:20), E321K (SEQ ID NO:22), D340R (SEQ ID NO:23), NO:24), D371R (SEQ ID NO:26), E438K (SEQ ID NO:28) and E440K (SEQ ID NO:30).

[0006] The present invention further includes T4 DNA ligase mutant amino acid sequences having at least one or more mutations, but wherein the remainder of the T4 DNA ligase mutant amino acid sequence has only conservative substitutions, such that the molecule has at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity with the corresponding T4 DNA ligase mutant amino acid sequence in the sequence listing (but without the tag sequence (SEQ ID NO: 32)) (hereinafter referred to as the "variant sequence").

[0007] The present invention further includes a DNA sequence identified by an odd-numbered sequence preceding each amino acid sequence of the above mutants (i.e., SEQ ID NOs: 3; 5; 7; 9; 11; 12; 13; 15; 17; 19; 21; 23; 25; 27; and 29, respectively, but without the 5' end portion of each sequence encoding a tag sequence), and further includes the aforementioned DNA sequences and other degenerate nucleic acid sequences (collectively referred to as "degenerate nucleic acid sequences"), which encode (i) each of the above T4 DNA ligase mutants, and (ii) each of the amino acid sequences of any one of the variant sequences.

[0008] The present invention further includes vectors incorporating any degenerate nucleic acid sequence; and cells transformed with any such vector or degenerate nucleic acid sequence and capable of expressing any of the above T4 DNA ligase mutant amino acid sequences or variant sequences.

[0009] The present invention also includes a composition or kit comprising any of the above T4 DNA ligase mutant amino acid sequences or variant sequences, degenerate nucleic acid sequences, or vectors incorporating such degenerate nucleic acid sequences. The present invention also includes a method for amplifying a target nucleic acid, wherein any of the above T4 DNA ligase mutant or variant sequences is employed in a reaction mixture designed to amplify the target nucleic acid, and the reagent mixture is subjected to conditions for amplifying the target nucleic acid.

[0010] The above mutant T4 DNA ligase mutants have greater activity at lower concentrations in amplifying target DNA sequences compared to the wild type, and the variant sequences are also expected to have this greater activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1A , Figure 1B A series of gel electrophoresis results of activity assays of each T4 DNA ligase mutant compared to wild-type T4 DNA ligase ("WT" in the upper left corner) are shown. There are 12 columns in each gel, so that from left to right, each column represents the concentration of T4 DNA ligase after 1:2 serial dilution, and wherein in column 1, 700ng of T4 DNA ligase (or mutant) are initially present in the solution. Each gel has an arrow at the dilution level, wherein a large amount of (upper band, marked as "1" on the right) supercoiled plasmid products and restricted digested linearized substrate plasmids (middle band, marked as "2" on the right) are clearly present. The bottom band (marked as "3" on the right) is an open-circular plasmid product connected by T4 DNA ligase wild-type or indicated mutants. DETAILED DESCRIPTION

[0012] The term "biologically active fragment" refers to any fragment, derivative, homolog or analog of a T4 DNA ligase mutant that has an in vivo or in vitro activity that is characteristic of a biomolecule; including, for example, ligase activity, or repairing mismatches present in nicked DNA by ligation. In some embodiments, a biologically active fragment, derivative, homolog or analog of a mutant T4 DNA ligase has any degree of biological activity of the mutant T4 DNA ligase in any in vivo or in vitro assay of interest.

[0013] In some embodiments, the biologically active fragment may optionally include any number of consecutive amino acid residues of a mutant T4 DNA ligase. The present invention also includes polynucleotides encoding any such biologically active fragments.

[0014] Biologically active fragments can be derived from translation of post-transcriptional processing or alternative splicing RNA, or alternatively can be produced by engineering, batch synthesis or other suitable operations. Biologically active fragments include fragments expressed in natural or endogenous cells, and fragments produced in expression systems (such as bacteria, yeast, plants, insects or mammalian cells).

[0015] As used herein, the phrase "conservative amino acid substitution" or "conservative mutation" refers to the replacement of one amino acid by another amino acid having common properties. A functional method for defining common properties between individual amino acids is to analyze the normalized frequency of amino acid changes between corresponding proteins of homologous organisms (Schulz (1979) Principles of Protein Structure, Springer-Verlag). According to such analysis, amino acid groups can be defined as groups in which amino acids preferentially exchange with each other and are therefore most similar to each other in terms of their effects on the overall protein structure (Schulz (1979) above). Examples of amino acid groups defined in this manner can include: "charged / polar group", including Glu, Asp, Asn, Gln, Lys, Arg and His; "aromatic or cyclic group", including Pro, Phe, Tyr and Trp; and "aliphatic group", including Gly, Ala, Val, Leu, Ile, Met, Ser, Thr and Cys. Within each group, subgroups can also be identified. For example, the group of charged / polar amino acids can be subdivided into multiple subgroups, including: a "positively charged subgroup" including Lys, Arg, and His; a "negatively charged subgroup" including Glu and Asp; and a "polar subgroup" including Asn and Gln. In another example, the aromatic or cyclic group can be subdivided into multiple subgroups, including: a "nitrogen ring subgroup" including Pro, His, and Trp; a "phenyl subgroup" including Phe and Tyr. In another further example, the aliphatic group can be subdivided into multiple subgroups, including: a "large aliphatic non-polar subgroup" including Val, Leu, and Ile; a "aliphatic weakly polar subgroup" including Met, Ser, Thr, and Cys; and a "small residue subgroup" including Gly and Ala. Examples of conservative mutations include amino acid substitutions of amino acids within the above subgroups, such as, but not limited to: Lys for Arg, and vice versa, so that a positive charge can be maintained; Glu for Asp, and vice versa, so that a negative charge can be maintained; Ser for Thr, and vice versa, so that a free -OH can be maintained; and Gln for Asn, and vice versa, so that a free -NH2 can be maintained. A "conservative variant" is a polypeptide that includes one or more amino acids that have been substituted to replace one or more amino acids of a reference polypeptide (e.g., a polypeptide whose sequence is disclosed in a publication or sequence database, or whose sequence has been determined by nucleic acid sequencing) with an amino acid having common properties (e.g., belonging to the same amino acid group or subgroup as described above).

[0016] When referring to a gene, "mutant" means that the gene has at least one base (nucleotide) change, deletion or insertion relative to the native or wild-type gene. The mutation (change, deletion and / or insertion of one or more nucleotides) can be in the coding region of the gene, or can be in an intron, 3'UTR, 5'UTR or promoter region. As a non-limiting example, a mutant gene can be a gene with an insertion in the promoter region, which can increase or decrease the expression of the gene; it can be a gene with a deletion, resulting in the production of a non-functional protein, a truncated protein, a dominant negative protein or no protein; or it can be a gene with one or more point mutations, resulting in an amino acid change in the encoded protein or resulting in abnormal splicing of the gene transcript.

[0017] The terms "mutant T4 DNA ligase of the present invention" and "mutant T4 DNA ligase" as used in this Detailed Description section, depending on the context, refer collectively or individually to mutant T4 DNA ligase polypeptides that have been tested and exhibit enhanced ligation activity, which are:

[0018] E23K (SEQ ID NO:4), E88K (SEQ ID NO:6), K98E (SEQ ID NO:8), E132K (SEQ ID NO:10), E143K (SEQ ID NO:12), E173K (SEQ ID NO:14), E240K (SEQ ID NO:16), E271K (SEQID NO:18), K306E (SEQ ID NO:20), E321K (SEQ ID NO:22), D340R (SEQ ID NO:24), D371R (SEQ ID NO:26), E438K (SEQ ID NO:28) and E440K (SEQ ID NO:30);

[0019] and / or variant sequences and / or degenerate nucleic acid sequences, as these terms are defined in the Summary of the Invention section.

[0020] "Naturally occurring" or "wild-type" refers to the form found in nature. For example, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence that is present in an organism and has not been intentionally modified by human manipulation.

[0021] The term "percent identity" or "homology" about nucleic acid or peptide sequences is defined as the percentage of nucleotides or amino acid residues identical to known polypeptides in a candidate sequence after the sequence is aligned to obtain the maximum percent identity and, if necessary, introducing a gap to achieve the maximum percent homology. N-terminal or C-terminal insertions or deletions should not be interpreted as affecting homology. Homology or identity at the nucleotide or amino acid sequence level can be determined by BLAST (Basic Local Alignment Search Tool) analysis, using the algorithm (Altschul (1997), Nucleic Acids Res. 25, 3389-3402 and Karlin (1990), Proc. Natl. Acad. Sci. USA 87, 2264-2268) used by programs blastp, blastn, blastx, tblastn and tblastx, which are customized for sequence similarity searches. The method used by BLAST program is to first consider the similar fragments (with or without gaps) between the query sequence and the database sequence, then evaluate the statistical significance of all matches identified, and finally summarize only those matches that meet the pre-selected significance threshold. For discussion of basic issues in sequence database similarity search, see Altschul (1994), Nature Genetics 6, 119-129. The search parameters of histogram, description, comparison, expectation (that is, for reporting the statistical significance threshold matched with the database sequence), cut-off, matrix and filter (low complexity) can be default settings. The default scoring matrix used by blastp, blastx, tblastn and tblastx is the BLOSUM62 matrix (Henikoff (1992), Proc.Natl.Acad.Sci.USA 89, 10915-10919), which is recommended for query sequences with a length exceeding 85 units (nucleotide bases or amino acids).

[0022] In some embodiments, the present invention relates to a method (and related kits, systems, apparatus and compositions) for performing a ligation reaction, the method comprising or consisting of: contacting a mutant T4 DNA ligase or a biologically active fragment thereof with a nucleic acid template in the presence of one or more nucleotides, and ligating at least one of the one or more nucleotides using the mutant T4 DNA ligase or a biologically active fragment thereof.

[0023] In some embodiments, the method may include ligating a double-stranded RNA or DNA polynucleotide chain into a circular molecule. In some embodiments, the method may further include detecting a signal indicative of ligation using a sensor. In some embodiments, the sensor is an ISFET. In some embodiments, the sensor may include a detectable label or detectable reagent in the ligation reaction.

[0024] In some embodiments, the present invention relates to methods (and related kits, systems, apparatus and compositions) for performing rolling circle amplification of nucleic acids (see U.S. Pat. No. 5,714,320, incorporated by reference), using a mutant T4 DNA ligase as an enzyme in the ligation step of the amplification process. Amplification includes amplifying nucleic acids in solution, and clonally amplifying nucleic acids on a solid support (such as nucleic acid beads, flow cells, nucleic acid arrays, or wells present on the surface of a solid support).

[0025] Preparation of mutant T4 DNA ligase

[0026] The mutant T4 DNA ligase of the invention can be expressed in any suitable host system, including bacterial, yeast, fungal, baculovirus, plant or mammalian host cells. For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present disclosure include promoters obtained from the E. coli lactose operon, the Streptomyces coelicolor agarase gene (dagA), the Bacillus subtilis levansucrase gene (sacB), the Bacillus licheniformis alpha-amylase gene (amyL), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), the Bacillus licheniformis penicillinase gene (penP), the Bacillus subtilis xylA and xylB genes, and prokaryotic β-lactamase genes (Villa-Kamaroff et al., 1978, Proc. Natl Acad. Sci. USA 75:3727-3731), and the tac promoter (DeBoer et al., 1983, Proc. Natl Acad. Sci. USA 80:21-25).

[0027] For filamentous fungal host cells, suitable promoters for directing transcription of the disclosed nucleic acid constructs include promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable α-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (WO 20 ). 96 / 00787), and the NA2-tpi promoter (a hybrid of the promoters from the Aspergillus niger neutral α-amylase and Aspergillus oryzae triose phosphate isomerase genes), and mutants, truncations and hybrid promoters thereof.

[0028] In yeast hosts, useful promoters can be derived from genes for the following enzymes: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8:423-488.

[0029] For baculovirus expression, insect cell lines from the order Lepidoptera (moths and butterflies), such as Spodoptera frugiperda, are used as hosts. Gene expression is controlled by a strong promoter (e.g., pPolh).

[0030] Plant expression vectors are based on the Ti plasmid of Agrobacterium tumefaciens, or on tobacco mosaic virus (TMV), potato virus X or cowpea mosaic virus. A commonly used constitutive promoter in plant expression vectors is the cauliflower mosaic virus (CaMV) 35S promoter.

[0031] For mammalian expression, cultured mammalian cell lines such as Chinese hamster ovary (CHO), COS, including human cell lines such as HEK and HeLa, can be used to produce mutant T4 DNA ligase. Examples of mammalian expression vectors include adenovirus vectors, pSV and pCMV series plasmid vectors, vaccinia virus and retroviral vectors, and baculovirus. Promoters of cytomegalovirus (CMV) and SV40 are commonly used in mammalian expression vectors to drive gene expression. Non-viral promoters, such as elongation factor (EF)-1 promoter are also known.

[0032] The control sequence for expression can also be a suitable transcription terminator sequence, that is, a sequence recognized by the host cell to terminate transcription. The terminator sequence is operably linked to the 3' end of the nucleic acid sequence encoding the polypeptide. Any terminator that is functional in the selected host cell can be used.

[0033] For example, exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

[0034] Exemplary terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase.

[0035] Terminators for insect, plant, and mammalian host cells are also well known.

[0036] The control sequence can also be a suitable leader sequence, i.e. the non-translational region of the mRNA that is very important for the translation performed by the host cell. The leader sequence can be operably linked to the 5' end of the nucleotide sequence encoding the polypeptide. Any leader sequence with functionality in the selected host cell can be used. The exemplary leader sequence for filamentous fungal host cells is obtained from the genes of Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase. The suitable leader sequence for yeast host cells is obtained from the following genes: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0037] The control sequence can also be a polyadenylation sequence, which is operably linked to the 3' end of the nucleic acid sequence and is recognized by the host cell as a signal for adding polyadenylic acid residues to the transcribed mRNA when transcribed. Any polyadenylation sequence that is functional in the selected host cell can be used in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells can be from the genes of the following enzymes: Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase.

[0038] The control sequence may also be a signal peptide coding region that encodes an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide to enter the secretory pathway of a cell. The 5' end of the coding sequence of the nucleic acid sequence may itself contain a signal peptide coding region that is naturally linked in the translation reading frame to the segment of the coding region that encodes the secreted polypeptide. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding region that is foreign to the coding sequence. In the case where the coding sequence does not naturally contain a signal peptide coding region, an exogenous signal peptide coding region may be required.

[0039] Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to enhance secretion of the polypeptide. However, any signal peptide coding region that directs the expressed polypeptide into the secretory pathway of a selected host cell may be used.

[0040] Effective signal peptide coding regions for bacterial host cells are those obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prs A. Further signal peptides are described by Simonen and Palva, 1993, Microbiol Rev 57: 109-137.

[0041] An effective signal peptide coding region for a filamentous fungal host cell can be the signal peptide coding region obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase.

[0042] Useful signal peptides for yeast host cells can be derived from the genes for Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae invertase.Signal peptides for other host cell systems are also well known.

[0043] The control sequence can also be a propeptide coding region encoding an amino acid sequence located at the amino terminus of a polypeptide. The resulting polypeptide is referred to as a proenzyme or propolypeptide (or in some cases as a zymogen). Propolypeptides are typically inactive and can be converted into mature active polypeptides by catalytic cleavage or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region can be obtained from the gene of the following enzyme: subtilis alkaline protease (aprE), subtilis neutral protease (nprT), saccharomyces cerevisiae α-factor, Rhizomucor miehei aspartic protease, and Myceliophthora thermophila lactase (WO 95 / 33836).

[0044] Where both the signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is located immediately adjacent to the amino terminus of the polypeptide and the signal peptide region is located immediately adjacent to the amino terminus of the propeptide region.

[0045] It may also be desirable to add regulatory sequences that allow expression of mutant T4 DNA ligase to be regulated relative to the growth of the host cell. Examples of regulatory systems are those systems that cause gene expression to be turned on or off in response to chemical or physical stimuli (including the presence of regulatory compounds). In prokaryotic host cells, suitable regulatory sequences include lac, tac, and trp operator systems. In yeast host cells, suitable regulatory systems include, for example, the ADH2 system or the GAL1 system. In filamentous fungi, suitable regulatory sequences include the TAKA alpha amylase promoter, the Aspergillus niger glucoamylase promoter, and the Aspergillus oryzae glucoamylase promoter. Regulatory systems for other host cells are also well known.

[0046] Other examples of regulatory sequences are those that allow gene amplification. In eukaryotic systems, these include dihydrofolate reductase genes that are amplified in the presence of methotrexate and metallothionein genes that are amplified with heavy metals. In these cases, the nucleic acid sequence encoding the KRED polypeptide of the invention will be operably linked to the regulatory sequence.

[0047] Another embodiment comprises a recombinant expression vector, which comprises a polynucleotide encoding an engineered mutant T4 DNA ligase or its variant, and one or more expression regulatory regions such as a promoter and terminator, and a replication origin, depending on the type of host into which they are introduced. The various nucleic acids and control sequences mentioned above can be linked together to produce a recombinant expression vector, which can include one or more convenient restriction sites to allow insertion or replacement of the nucleotide sequence encoding the mutant T4 DNA ligase at such sites. Alternatively, the nucleotide sequence of the mutant T4 DNA ligase can be expressed by inserting the nucleotide sequence or a nucleic acid construct comprising the sequence into a suitable vector for expression. When producing the expression vector, the encoding sequence is located in the vector so that the encoding sequence is operably connected with the suitable control sequence for expression.

[0048] The recombinant expression vector can be any vector (e.g., plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can cause expression of the mutant T4 DNA ligase polynucleotide sequence. The choice of vector will generally depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear plasmid or a closed circular plasmid.

[0049] The expression vector can be an autonomously replicating vector, i.e., a vector existing as an extrachromosomal entity, whose replication is independent of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome or an artificial chromosome. The vector can contain any means for ensuring self-replication. Alternatively, the vector can be a vector that is integrated into the genome and replicated together with one or more chromosomes into which it has been integrated when it is introduced into the host cell. In addition, a single vector or plasmid or two or more vectors or plasmids containing the total DNA to be introduced into the host cell genome together can be used, or a transposon can be used.

[0050] The expression vector herein preferably contains one or more selective markers, which allow for easy selection of transformed cells. A selective marker is a gene whose product provides biocide resistance or viral resistance, resistance to heavy metals, prototrophy to auxotrophic types, etc. Examples of bacterial selective markers are dal genes from Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance (such as ampicillin, kanamycin, chloramphenicol (Example 1) or tetracycline resistance). Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenylyltransferase), and trpC (anthranilate synthase), and equivalents thereof. Embodiments for use in Aspergillus cells include the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae, and the bar gene of Streptomyces hygroscopicus. Selectable markers for use in insect, plant, and mammalian cells are also well known.

[0051] The expression vector of the present invention preferably contains one or more elements that allow the vector to be integrated into the genome of the host cell or to replicate the vector autonomously in the cell independently of the genome. In order to be integrated into the host cell genome, the vector may rely on the nucleic acid sequence encoding the polypeptide or any other element of the vector to integrate the vector into the genome by homologous or non-homologous recombination.

[0052] Alternatively, the expression vector can contain other nucleotide sequences for guiding integration into the genome of the host cell by homologous recombination. Other nucleotide sequences enable the vector to be integrated into one or more precise locations of one or more chromosomes in the host cell genome. Integration elements can be any sequence homologous to the target sequence in the host cell genome. In addition, integration elements can be non-coding or encoding nucleic acid sequences. On the other hand, the vector can be integrated into the genome of the host cell by non-homologous recombination.

[0053] For autonomous replication, the vector may further include an origin of replication that enables the vector to autonomously replicate in the host cell. The example of a bacterial origin of replication is P15A ori, or the origin of replication of plasmid pBR322, pUC19, pACYC177 (the plasmid has P15A ori) or pACYC184 allowed to be replicated in Escherichia coli, and the origin of replication of plasmid pUB110, pE194, pTA1060 or pAM31 allowed to be replicated in bacillus. The example of the origin of replication for yeast host cells is a combination of 2 microns of origin of replication, ARS1, ARS4, ARS1 and CEN3, and ARS4 and CEN6. The origin of replication can be a replication origin with a mutation that makes its function in the host cell sensitive to temperature (see, e.g., Ehrlich, 1978, Proc Natl Acad Sci. USA 75:1433).

[0054] More than one copy of the nucleic acid sequence of the mutant T4 DNA ligase may be inserted into the host cell to increase the production of the gene product. An increased copy number of the nucleic acid sequence may be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the nucleic acid sequence, wherein cells containing amplified copies of the selectable marker gene, and thus additional copies of the nucleic acid sequence, may be selected by culturing the cells in the presence of an appropriate selective agent.

[0055] Expression vectors for mutant T4 DNA ligase polynucleotides are commercially available. Suitable commercial expression vectors include p3xFLAGTM expression vectors from Sigma-Aldrich Chemicals in St. Louis, Missouri, which include a CMV promoter and hGH polyadenylation site for expression in mammalian host cells, and a pBR322 origin of replication and an ampicillin resistance marker for amplification in E. coli. Suitable other expression vectors are pBluescriptII SK(-) and pBK-CMV commercially available from Stratagene in La Jolla, California, and plasmids derived from pBR322 (Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4 (Invitrogen), or pPoly (Lathe et al., 1987, Gene 57:193-201).

[0056] Suitable host cells for expressing polynucleotides encoding mutant T4 DNA ligase are well known in the art, and include, but are not limited to, bacterial cells such as Escherichia coli, Lactobacillus kefir, Lactobacillus brevis, Lactobacillus minor, Streptomyces, and Salmonella typhimurium cells; fungal cells such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; and plant cells. Suitable culture media and growth conditions for the above host cells are well known in the art.

[0057] The polynucleotides for expressing mutant T4 DNA ligase can be introduced into cells by various methods known in the art. These techniques include electroporation, biolistic particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion. Various methods for introducing polynucleotides into cells are known to the skilled artisan.

[0058] The polynucleotide encoding mutant T4 DNA ligase can be prepared by standard solid phase methods according to known synthetic methods. In some embodiments, fragments of up to about 100 bases can be synthesized separately, and then connected (for example, by enzymatic or chemical ligation methods, or polymerase-mediated methods) to form any desired continuous sequence. For example, the classical phosphoramidite method described by Beaucage et al., 1981, Tet Lett 22: 1859-69, or the method described by Matthes et al., 1984, EMBO J. 3: 801-05 (for example, as it is generally applied in automatic synthesis methods) can be used to prepare polynucleotides by chemical synthesis. According to the phosphoramidite method, for example, oligonucleotides are synthesized in an automatic DNA synthesizer, purified, annealed, connected and cloned into a suitable vector. In addition, essentially any nucleic acid can be obtained from a variety of commercial sources, such as The Midland Certified Reagent Company of Midland, Texas, The Great American Gene Company of Ramona, California, ExpressGen Inc. of Chicago, Illinois, and Operon Technologies Inc. of Alameda, California.

[0059] Engineered mutant T4 DNA ligase expressed in host cells can be recovered from cells and / or culture medium using any one or more of the well-known protein purification techniques, including lysozyme treatment, sonication, filtration, salting out, ultracentrifugation, and chromatography. Suitable solutions for lysis and efficient protein extraction from bacteria such as E. coli are commercially available from Sigma-Aldrich of St. Louis under the trade name CelLytic B.TM.

[0060] Chromatographic techniques used to isolate mutant T4 DNA ligase include reverse phase chromatography, high performance liquid chromatography, ion exchange chromatography, gel electrophoresis and affinity chromatography. Purification conditions will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, and will be apparent to those skilled in the art.

[0061] In some embodiments, affinity techniques can be used to separate mutant T4 DNA ligase. For affinity chromatography purification, any antibody that specifically binds to mutant T4 DNA ligase can be used. In order to produce antibodies, various host animals (including but not limited to rabbits, mice, rats, etc.) can be immunized by injection of compounds. The compound can be attached to a suitable carrier, such as BSA, through a side chain functional group or a linker attached to a side chain functional group. Various adjuvants can be used to enhance the immune response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, complex polyols (pluronic polyols), polyanions, peptides, oil emulsions, keyhole limpet hemocyanin (keyhole limpet hemocyanin), dinitrophenol, and potentially useful human adjuvants such as BCG (BCG) and Corynebacterium parvum (Corynebacterium parvum). Examples of preparing T4 DNA ligase mutants

[0062] T4 DNA ligase mutants were generated by conventional PCR mutagenesis, where primers were designed to contain the desired base substitutions and during the PCR the mutations were incorporated into the amplicon, replacing the original sequence. All T4 DNA ligase mutants and the wild type have a C-terminal 6-mer His tag added to facilitate purification, preceded by a 6-mer series of Ser and Gly residues as indicated.

[0063] PCR is followed by Dpnl digestion, which destroys the methylated template (containing no substitutions), leaving only the unmethylated PCR amplicon with the substitutions.

[0064] The PCR amplicon is then transformed directly into chemically competent E. coli host cells, where the bacteria are pre-treated with chemicals to enable them to take up the plasmid into which the amplicon has been incorporated. See ThermoFisher Scientific, Chemically Competent Cells webpage (kits for generating chemically competent cells are provided).

[0065] Mutant T4 DNA ligase polypeptides expressed by transformed E. coli host cells were characterized and selected based on standard ligation assays with gel electrophoresis. Ligase catalyzes the formation of phosphodiester bonds between the 5' and 3' ends of complementary cohesive ends or blunt ends of double-stranded DNA, and the degree of connection with different T4 DNA ligase mutants can be visualized on agarose gels using appropriate DNA dyes. In this case, GelRed (Biotium, San Francisco, California) was used to dye the gel so that it can be visualized under UV light. The performance of each T4 DNA ligase mutant was checked according to its ligation activity under reduced enzyme concentrations, and the activity of the resulting wild-type (" WT ") ligase was compared with the activity of a similarly diluted wild-type, allowing determination of which mutants showed increased activity compared to the wild-type under the same conditions.

[0066] Ligation substrates for characterization of mutant T4 DNA ligase polypeptides expressed by transformed E. coli host cells were prepared as follows.

[0067] The DNA vector used was pUC19 (New England Biolabs, catalog number N3041S). PUC19 is a double-stranded circle 2686 base pairs long. (New England Biolabs, catalog number R3733S) digestion of pUC19, where cleavage on the 5' strand occurs after the addition of one random (N1) nucleotide to the recognition sequence, and cleavage on the 3' strand occurs after the addition of five additional random (N5) nucleotides to the complementary recognition sequence. The 5' recognition sequence is GGTCTC (SEQ ID NO: 31). The cleavage site for pUC19 was designated 5′-GGTCTC(N1) / (N5)-3′.

[0068] Mix 5 μl of 1 mg / ml puC19 with 2.5 μl of 20,000 units / ml 5μl 10XrCutSmart TMBuffer (New England Biolabs, catalog number B6004S) (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg / ml recombinant albumin) and 35 μl of water were combined. After all components were combined, the composition was incubated at 37°C for digestion. After 1 hour at 37°C, the reaction was incubated at 80°C for 20 minutes to allow Heat inactivation. The mixture was then diluted with water to a concentration of 10 ng / μl.

[0069] Example: Ligation Assay and Results

[0070] The ligation procedure was performed as follows. Each T4 DNA ligase (whether wild type or variant) was diluted with enzyme diluent (50% glycerol, 10mM tris-HCl) under serial dilution so that 10 different concentrations were obtained for each sample. The starting concentration of each serial dilution was 100ng / ul, which was diluted twice in each next dilution so that the final concentration was 50% of the sample concentration before dilution, and so on, for a total of 10 1:2 dilutions. Then 7μl of each enzyme or serial dilution was added to the PCR plate so that the approximate amount of enzyme in lanes 1 to 10 was 700, 350, 175, 88, 44, 22, 11, 5.5, 2.7, 1.4ng, respectively. Lanes 11 and 12 did not add mutations or wild type T4 DNA ligase to the reaction, showing how far the reaction was carried out when no connection occurred.

[0071] For the enzyme, 13 μl of 2 μl of 10X T4 DNA ligase reaction buffer (New England Biolabs, catalog number B0202A; including 50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT (dithiothreitol)), 1 μl of 10 ng / μl A master mix consisting of digested pUC19 and 10 μl of water was added to each reaction. This brought the total volume of each reaction to 23 μl. The reactions were incubated at 16°C for 30 minutes. After the 30-minute incubation period, the reactions were subjected to a heat shock at 80°C for 2 minutes to stop any further activity. 4 μl of stop solution (120 mM EDTA, 30% glycerol, 50 mM Tris-HCl pH 8.0, 0.0125% bromophenol blue, 0.1% SDS, and 5x Gel Red Nucleic Acid Stain (Biotium, Fremont, CA) was added to each reaction.

[0072] Gel electrophoresis with 8% agarose gel was used to visualize the ligation reaction products. Each gel had a wild-type T4 DNA ligase sample set and a 7 variant T4 DNA ligase sample set. Each gel was run at 200V for 25 minutes.

[0073] The results compared with the wild type Figure 1A , 1B The identified mutants that exhibited increased ligation activity were as follows: E23K, E88K, K98E, E132K, E143K, E173K, E240K E271K, K306E, E321K, D340R, D371R, E438K and E440K. Figure 1A , 1B shown.

[0074] The comparative activity results are listed in Table 1 below, showing the lane differences for each T4 DNA ligase mutant, where each lane difference greater than WT is assigned a 2-fold increase in activity. For example, a 1-lane activity improvement relative to WT is assigned a value of 2 (as indicated by little or no apparent upper band representing supercoiled plasmid product and little or no apparent middle band representing restriction digested linear substrate plasmid in the lane), while a 2-lane activity improvement relative to WT is assigned a value of 4, and so on.

[0075] Table 1: T4 DNA ligase variants with greater activity than WT

[0076]

[0077]

[0078] The specific methods and compositions described herein are representative of preferred embodiments, and are exemplary and are not intended to limit the scope of the invention. In view of this specification, other purposes, aspects and embodiments will occur to those skilled in the art, and are included in the spirit of the invention as defined by the scope of the claims. It will be apparent to those skilled in the art that various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention described illustratively herein may be appropriately practiced in the absence of any one or more elements, or any one or more restrictions, that are not explicitly disclosed herein as necessary. Thus, for example, in each case herein, in the embodiments or examples of the present invention, any of the terms "comprising", "including", "containing", etc. should be read broadly and without restriction. The methods and processes exemplarily described herein may be appropriately implemented in different step orders, and they are not necessarily limited to the step orders indicated herein or in the claims. It should also be noted that, unless the context clearly indicates otherwise, as used herein and in the appended claims, the singular forms "one / a kind" and "said" include plural indicators, and the plural includes the singular form. In any case, this patent application cannot be interpreted as being limited to the specific examples or embodiments or methods specifically disclosed herein.

[0079] The present invention has been described broadly and generally herein. Each of the narrower species and subgeneric classifications falling within the total disclosure also forms a part of the present invention. The terms and expressions that have been adopted are used as terms of description and are not restrictive, and are not intended to exclude any equivalents or portions thereof of the features shown and described using such terms and expressions, but it will be appreciated that different modifications within the scope of the invention claimed are possible. Therefore, it should be understood that, although the present invention has been specifically disclosed by preferred embodiments and optional features, modifications and changes of the concepts disclosed herein may be adopted by those skilled in the art, including but not limited to variant sequences, and such modifications and changes are considered to be within the scope of the invention defined by the appended claims.

Claims

1. A mutant T4 DNA ligase, the amino acid sequence of which is shown in SEQ ID NO: 12 or 14.

2. A mutant T4 DNA ligase, whose amino acid sequence is shown in SEQ ID NO: 12 or 14, excluding its 6-membered histidine tag at the C-terminus and the preceding 8 glycine, leucine and serine amino acid residues.

3. A polynucleotide encoding the amino acid sequence of any one of the mutant T4 DNA ligases as claimed in claim 1.

4. A polynucleotide encoding the amino acid sequence of any one of the mutant T4 DNA ligases as claimed in claim 2.

5. A polynucleotide as claimed in claim 3, whose DNA sequence is shown in SEQ ID NO:11 or 13. A vector comprising the polynucleotide according to any one of claims 3 to 5.

7. A cell transformed with the polynucleotide according to any one of claims 3 to 5 and expressing the polynucleotide according to any one of claims 3 to 5; the cell is not an animal cell or a plant cell.

8. A method for performing polynucleotide ligation between different polynucleotides or performing polynucleotide ligation by ligating the 5' end and the 3' end of the polynucleotides to produce a circular polynucleotide, wherein the polynucleotides have blunt ends or sticky ends, the method comprising: Providing a ligation mixture comprising the polynucleotides to be ligated and the mutant T4 DNA ligase according to claim 2; and The ligation mixture is exposed to a temperature at which ligation occurs.

9. A method for performing polynucleotide ligation between different polynucleotides or performing polynucleotide ligation by ligating the 5' end and the 3' end of the polynucleotides to produce a circular polynucleotide, wherein the polynucleotides have blunt ends or sticky ends, the method comprising: Providing a ligation mixture comprising a polynucleotide to be ligated and the mutant T4 DNA ligase according to claim 1; and The ligation mixture is exposed to a temperature at which ligation occurs.

10. The method of claim 8 or 9, wherein the ligation mixture further comprises Tris-HCl, MgCl2, ATP, dithiothreitol and water.

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