Positioning fusion functional protein, chassis cell and application of positioning fusion functional protein and chassis cell

Abstract

The invention provides a positioning fusion functional protein, a chassis cell and application of the positioning fusion functional protein and the chassis cell, and belongs to the technical field of biological medicine. According to the invention, a positioning fusion functional protein is firstly constructed, a chassis cell is constructed based on the positioning fusion functional protein, and the chassis cell can be specifically applied to repeated tandem expression of recombinant micromolecular collagen; according to the invention, in a protein secretory expression pathway, Kex2 enzyme digestion recognition site dual-basic amino acids connected with tandem monomers and specific amino acids added before and after the dual-basic amino acids and used for improving Kex2 recognition cutting efficiency can be completely removed, so that recombinant micromolecular collagen which is 100% consistent with a natural human collagen sequence is obtained; the Kex2 enzyme digestion efficiency is guaranteed, meanwhile, the introduced amino acid is not necessarily collagen amino acid, the design space is greatly enlarged, and the method has good practicability.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a localized fusion functional protein, chassis cells and uses thereof. Background Art

[0002] Collagen has biological properties such as low immunogenicity, good biocompatibility, and cell growth promotion, and is widely used in biomedicine, beauty and skin care and other fields. With the increasing market demand for collagen transdermal absorption, small molecule collagen has gradually become a research hotspot in recent years. Existing small molecule collagen is mostly a mixture of small molecule collagen peptides obtained by hydrolysis and enzymatic hydrolysis of animal-derived collagen or by heat treatment of recombinant collagen. It has problems such as unclear amino acid sequence and molecular weight distribution, and poor batch reproducibility. The use of genetic engineering technology to prepare recombinant small molecule collagen can effectively solve the above problems.

[0003] There are few studies on recombinant small molecule collagen. Some researchers have expressed a variety of recombinant small molecule collagen using the Pichia pastoris expression system. They designed a tandem repeat expression sequence and constructed an engineered strain that expresses a localized fusion functional protein, so that the tandem sequence is cut into target monomers during cell secretion. Although this technical solution obtains a small molecule collagen with a single and clear amino acid sequence and molecular weight, and greatly improves the yield of single-sequence recombinant small molecule collagen in the eukaryotic expression system, it also avoids the cost and risk of exogenous protein residues caused by in vitro protease cleavage. However, the recombinant small molecule collagen obtained by this method still has the problem that the amino acid at the P3 position (the endogenous protease Kex2 of Pichia pastoris cuts at the C-terminus of the dibasic amino acid KR, the upstream of the cleavage site is numbered Pn, the downstream is numbered Pn', and n increases in the direction away from the cleavage site, for example: R is P1, K is P2, and so on) cannot be removed, and the definition of Class A recombinant humanized collagen in the "Guidelines for the Naming of Recombinant Collagen Biomaterials" requires that it does not contain non-human collagen amino acid sequences. The amino acids in the residual non-natural collagen sequences make it impossible to meet the requirements of humanized collagen, and the immunogenicity cannot be guaranteed.

[0004] Therefore, a method needs to be provided to solve the problem that the residual restriction sites after repeated tandem expression of recombinant small molecule collagen in Pichia pastoris and the non-human collagen sequence amino acid residues added before and after the restriction recognition sites to improve the restriction efficiency cannot be completely removed. Summary of the invention

[0005] In view of some deficiencies in the prior art, the present invention provides a localized fusion functional protein, a chassis cell and uses thereof; the present invention first constructs a localized fusion functional protein, and constructs a chassis cell based on the localized fusion functional protein, the chassis cell can be specifically applied to the repeated tandem expression of recombinant small molecule collagen, can completely remove the double basic amino acids of the Kex2 enzyme cleavage recognition site connecting the tandem monomers and the specific amino acids added before and after the double basic amino acids for improving the Kex2 recognition and cleavage efficiency in the protein secretion expression pathway, and obtain a recombinant small molecule collagen that is 100% consistent with the natural human collagen sequence. In the process of designing the repeated tandem sequence, the Kex2 enzyme cleavage efficiency is guaranteed while the introduced amino acids do not necessarily have to be the collagen's own amino acids, which greatly improves the design space and has good practicality.

[0006] In order to achieve the above technical objectives, the present invention adopts the following technical means:

[0007] The present invention first provides a localization fusion functional protein, which comprises carboxypeptidase A (CPA) and a functional region having intracellular membrane localization or conversion and transport functions between organelles.

[0008] Preferably, the localization fusion functional protein further comprises a connection sequence, and the connection sequence is used for connection when carboxypeptidase A is fused with a functional region sequence having intracellular membrane localization or conversion and transport functions between organelles for expression.

[0009] Preferably, the connection sequence is a Linker sequence, as shown in SEQ ID NO.5.

[0010] Preferably, the functional region sequence having the function of intracellular membrane localization or conversion and transport between organelles is derived from Kex2 of Saccharomyces cerevisiae or Pichia pastoris, or other protein functional regions with similar functions such as Ste13 protease;

[0011] The sequence of the functional region having the function of intracellular membrane localization or conversion and transport between organelles is shown in SEQ ID NO:4.

[0012] Preferably, the carboxypeptidase A localization fusion functional protein is fused to an α-factor secretion signal signal peptide sequence at its N-terminus and to a myc tag at its C-terminus.

[0013] Preferably, the amino acid sequence of the carboxypeptidase A localization fusion functional protein includes SEQ ID NO: 1 or an amino acid sequence having more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% identity with SEQ ID NO: 1.

[0014] The present invention also provides a nucleic acid, which encodes the above-mentioned carboxypeptidase A localization fusion functional protein.

[0015] Preferably, the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO: 2, or a degenerate sequence thereof.

[0016] The present invention also provides a recombinant expression vector comprising the nucleic acid.

[0017] Preferably, the method for constructing the expression recombinant vector comprises:

[0018] The nucleic acid encoding the localization fusion functional protein is cloned into plasmid 1 to obtain recombinant plasmid 1, and the recombinant plasmid 1 is assembled with plasmid 2 to obtain recombinant plasmid 2;

[0019] The recombinant plasmid 2, the DAS1 promoter and the AOX terminator are cloned into the plasmid 3 to obtain the recombinant expression vector.

[0020] Preferably, the plasmid 1 includes pUC57-BasI-Free; the plasmid 2 includes BB1_23; and the plasmid 3 includes BB3eH_14.

[0021] The present invention also provides a chassis cell comprising the nucleic acid, or comprising the recombinant vector, or expressing the above-mentioned localized fusion functional protein.

[0022] Preferably, the host bacteria of the chassis cells include Pichia pastoris, Saccharomyces cerevisiae, Hansenula, or bacteria expressing carboxypeptidase B (CPB) localization fusion functional protein, etc., preferably Pichia pastoris.

[0023] Preferably, the bacteria expressing carboxypeptidase B localization fusion functional protein include GS115 / HCPB and X33 / HCPB.

[0024] According to an embodiment of the present invention, the chassis cells are deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration, with the deposit numbers being CGMCC No.29610 and CGMCC No.29609.

[0025] Preferably, the chassis cells also simultaneously express carboxypeptidase B localization fusion functional protein.

[0026] According to an embodiment of the present invention, the chassis cells are deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration, with the deposit numbers being CGMCC No.29607 and CGMCC No.29608.

[0027] The invention also discloses the use of the chassis cells in expressing recombinant small molecule collagen.

[0028] The present invention also discloses a method for obtaining recombinant small molecule collagen, the method comprising:

[0029] The tandem repeat expression sequence of the recombinant small molecule collagen is connected to the expression vector and then transferred into the chassis cells to obtain the recombinant engineering bacteria containing or expressing the recombinant small molecule collagen, and the expression is induced by fermentation to obtain the recombinant small molecule collagen. Compared with the prior art, the beneficial effects of the present invention are:

[0030] The present invention first constructs a localization fusion functional protein, and constructs a chassis cell based on the localization fusion functional protein. The chassis cell can be specifically used for repeated tandem expression of recombinant small molecule collagen, and can completely remove the double basic amino acids of the Kex2 enzyme cleavage recognition site connecting the tandem monomers and the specific amino acids added before and after the double basic amino acids for improving the Kex2 recognition and cleavage efficiency in the protein secretion expression pathway, so as to obtain a recombinant small molecule collagen that is 100% consistent with the natural human collagen sequence. In the process of designing the repeated tandem sequence, the Kex2 enzyme cleavage efficiency is guaranteed, and the introduced amino acids do not have to be the collagen's own amino acids, which greatly improves the design space and has good practicality.

[0031] The chassis cells of the present invention can be used for tandem expression of recombinant small molecule collagen to obtain recombinant small molecule collagen. This method can solve the problem of residual enzyme cleavage sites after repeated tandem expression of recombinant small molecule collagen in Pichia pastoris and the problem that the non-human collagen sequence amino acid residues added before and after the enzyme cleavage recognition sites for improving the enzyme cleavage efficiency cannot be completely removed.

[0032] In the carboxypeptidase A localization fusion functional protein of the present invention, carboxypeptidase A can remove amino acid residues other than D, E, R, K, and P at the C-terminus, and prefers hydrophobic amino acids. At the same time, other amino acids in the peptide have an important influence on its cutting specificity, and Pro and Gly at the P1 site also have an inhibitory effect on its cutting. The protein can effectively solve the problem that the residual enzyme cutting sites after the recombinant small molecule collagen protein is repeatedly expressed in series in Pichia pastoris and the non-human collagen sequence amino acid residues added before and after the enzyme cutting recognition site for improving the enzyme cutting efficiency cannot be completely removed. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The electrophoresis bands were verified by colony PCR.

[0034] Figure 2Western Blot detection results of Anti-Myc (a), Anti-Carboxypeptidase A (b), and rabbit anti-CPB1 polyclonal antibody (c).

[0035] Figure 3 The SDS-PAGE electrophoresis diagram of samples COL3LF and COL3LV.

[0036] Figure 4 The secondary mass spectra of samples COL3LF (a) and COL3LV (b) DETAILED DESCRIPTION

[0037] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto. In the embodiments of the present invention, all that is not described in detail is accomplished by conventional experimental methods, and all that is not described in detail in the processes involved in the embodiments is understandable and easily implemented by those skilled in the art based on the product manual or basic knowledge in the art, and therefore will not be described in detail.

[0038] Unless otherwise specified, all culture media or other reagent materials involved in the present invention are conventionally prepared or purchased.

[0039] Example 1: Obtaining the recombinant expression vector BB3eH-CPA-PPKEX2

[0040] (1) Design of carboxypeptidase A localization fusion functional protein:

[0041] The amino acids 111-419 (SEQ ID NO: 3) in the sequence of human carboxypeptidase A (https: / / www.uniprot.org / uniprotkb / P15085) and the amino acids 681-777 (SEQ ID NO: 4) in the sequence of fusion-expressed Pichia pastoris Kex2 (https: / / www.uniprot.org / uniprotkb / C4R095) were selected, and the SEQ ID NO: 3 sequence and the SEQ ID NO: 4 sequence were connected using Linker (SEQ ID NO: 5), and then the α-factor secretion signal signal peptide sequence was fused to the N-terminus and the myc tag was fused to the C-terminus to obtain the carboxypeptidase A localization fusion functional protein, named CPA-PPKEX2, whose amino acid sequence is shown in SEQ ID NO: 1, and the nucleotide sequence encoding CPA-PPKEX2 is shown in SEQ ID NO: 2.

[0042] SEQ ID NO: 1:

[0043] MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKRARSTDTFNYATYHTLEEIYDFLDLLVAENPHLVSKIQIGNTYEGRPIYVLKFSTGGSKRPAIWIDTGIHSREWVTQASGVWFAKKITQDYGQDAAFTAILDTLDIFLEIVTNPDGFAFTHSTNRMWRKTRSHTAGSLCIGVDPNRNWDAGFGLSGASSNPCSETYHGKFANSEVEVKSIVDFVKDHGNIKAFISIHSYSQLLMYPYGYKTEPVPDQDELDQLSKAAVTALASLYGTKFNYGSIIKAIYQASGSTIDWTYSQGIKYSFTFELRDTGRYGFLLPASQIIPTAKETWLALLTIMEHTLNHPYGGSGSGSGGSYLAFLLGLGFLICIIFLFTNRNKLERRQRRNRRDEYEFDLIPADDDFDTEEDQEANSQFTLDSDAELMFEDTSQREASPHEYQDSLGSNEHPKRAALEQKLISEEDL

[0044] SEQ ID NO:2:

[0045]

[0046] SEQ ID NO:3:

[0047] ARSTDTFNYATYHTLEEIYDFLDLLVAENPHLVSKIQIGNTYEGRPIYVLKFSTGGSKRPAIWI

[0048] DTGIHSREWVTQASGVWFAKKITQDYGQDAAFTAILDTLDIFLEIVTNPDGFAFTHSTNRM

[0049] WRKTRSHTAGSLCIGVDPNRNWDAGFGLSGASSNPCSETYHGKFANSEVEVKSIVDFVKD

[0050] HGNIKAFISIHSYSQLLMYPYGYKTEPVPDQDELDQLSKAAVTALASLYGTKFNYGSIIKAIY

[0051] QASGSTIDWTYSQGIKYSFTFELRDTGRYGFLLLPASQIIPTAKETWLALLTIMEHTLNHPY

[0052] SEQ ID NO:4:

[0053] YLAFLLGLGFLICIIFLFTNRNKLERRQRRNRRDEYEFDLIPADDDFDTEEDQEANSQFTLDSDAELMFEDTSQREASPHEYQDSLGSNEHPKRAAL

[0054] SEQ ID NO:5:

[0055] GGSGSGSGGS

[0056] (2) Construction of recombinant expression vector:

[0057] Nanjing GenScript Biotechnology Co., Ltd. was commissioned to synthesize the sequence SEQ ID NO: 2, and then SEQ ID NO: 2 was cloned into the vector pUC57-BasI-Free (purchased from Nanjing GenScript Biotechnology Co., Ltd.) to obtain the plasmid

[0058] 2_MFα-CPA1(chain)-pPKEX2_pUC57-BsaI-free.

[0059] By BsaI- The above plasmids were cloned into plasmid BB1_23 (Addgene, Cat. No.: 39296) using the v2 kit (purchased from NEB), with the amount of each plasmid being 50 fmol. After reacting at 37°C for 5 min, the temperature was raised to 60°C for 5 min. After the reaction, the assembly product, i.e., BB1_23_CPA, was obtained. 10 μL of the assembly product was transformed into DH5α E. coli competent cells, and then the transformation product was coated on an LB plate containing 50 μg / mL Kan, cultured overnight in a 37°C incubator, and a single colony was picked and inoculated into 5 mL of LB culture medium, cultured overnight at 37°C and 220 rpm, and the plasmid was extracted with a plasmid mini-extraction kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.) to obtain plasmid BB1_23_CPA.

[0060] Plasmid BB1_12_pDAS1 (addgene, catalog number: 39296), plasmid BB1_34_AOXtt and plasmid BB1_23_CPA were cloned into plasmid BB3eH_14 (addgene, catalog number: 39296) using FastDigest BpiI and T4 DNA Ligase (both purchased from Thermo Fisher Scientific). The amount of each plasmid was 30 fmol, and the reaction conditions were as follows: react at 37°C for 1 min, then cool to 16°C for 1 min, cycle 30 times, then heat to 60°C for 5 min, and after the reaction, the assembly product was obtained, namely the recombinant expression vector BB3eH-CPA-PPKEX2. 10 μL of the assembly product was transformed into DH5α Escherichia coli competent cells, and the transformation product was spread on an LB plate containing 50 μg / mL HygR, cultured overnight in a 37°C incubator, and a single colony was picked and inoculated into 5 mL LB medium, cultured overnight at 37°C and 220 rpm, and the plasmid was extracted with a plasmid extraction kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.), and the extracted plasmid was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. After verification, the recombinant expression vector BB3eH-CPA-PPKEX2 was obtained.

[0061] Example 2: Construction of a recombinant engineered strain containing carboxypeptidase A localization fusion functional protein

[0062] (1) Construction and screening of recombinant engineering strains:

[0063] 10 μg of the recombinant expression vector BB3eH-CPA-PPKEX2 constructed in Example 1 was digested with PmeI (purchased from NEB) at 37°C overnight according to the kit instructions to linearize it, and then the linearized plasmid was recovered using a PCR product purification kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.).

[0064] Take 10 μL of linearized plasmid and electrotransform into Pichia GS115 (purchased from Thermo Fisher Scientific), X33 (purchased from Thermo Fisher Scientific), GS115 / HCPB (disclosed in the patent with publication number CN116948013A, strain deposit number CGMCCNo.25815), X33 / HCPB (disclosed in the patent with publication number CN116948013A, plasmid pPICZαB-HCPB-PPKEX2 electrotransformed into Pichia X33) competent cells. After the transformation, take 200 μL of the transformation product and spread it on a YPD plate containing 300 μg / mL HygR, and invert and culture it in a 30°C incubator for 3 to 4 days until a single colony appears.

[0065] Pick a single colony from the YPD plate into a sterile 96-well plate (200μL YPD / well), mix well and culture at 30℃ for 48h. After the culture, take 50μL of the bacterial solution and centrifuge it at 4000rpm for 5min in a PCR well plate. After centrifugation, discard the supernatant, wash the bacteria with 100μL water, and then centrifuge it at 4000rpm for 5min. After centrifugation, discard the supernatant, add 10μL 0.02M NaOH solution and vortex to suspend the bacteria. Incubate it at 99℃ in a PCR instrument for 10min before taking it out as a PCR template.

[0066] Veri-F and Veri-R were used as primers, and Premix Taq enzyme (TaKaRa Taq Version 2.0 plus dye) was used for colony PCR verification. The PCR program was (94°C, 30s→55°C, 30s→72°C, 2'30s)×30. 5'AOX and 3'AOX primers were used for PCR as positive controls. 10 μL of the reaction solution was used for agarose electrophoresis verification. The results were as follows: Figure 1 As shown. Figure 1 As can be seen, bands of expected sizes were obtained (Veri-F / Veri-R 972bp; 5'AOX / 3'AOX 2200bp), indicating that the target gene was successfully introduced into GS115, X33, GS115 / HCPB and X33 / HCPB, namely, chassis cells CPA / GS115 and CPA / X33 expressing carboxypeptidase A localization fusion functional protein, and chassis cells CPA / HCPB-GS115 and CPA / HCPB-X33 co-expressing carboxypeptidase A and carboxypeptidase B localization fusion functional proteins were obtained.

[0067] Veri-F:TCCAGCAGACGACGACTTCG (SEQ ID NO:6);

[0068] Veri-R: CAGGACATATCCACGCCCT (SEQ ID NO:7);

[0069] 5'AOX: GACTGGTTCCAATTGACAAGC (SEQ ID NO:8);

[0070] 3'AOX: GCAAATGGCATTCTGACATCC (SEQ ID NO:9).

[0071] The chassis cells CPA / GS115 and CPA / X33 expressing carboxypeptidase A localization fusion functional protein, and the chassis cells CPA / HCPB-GS115 and CPA / HCPB-X33 co-expressing carboxypeptidase A and carboxypeptidase B localization fusion functional protein were sent to the General Microbiological Center of China Microbiological Culture Collection Administration for preservation. The chassis cell preservation numbers correspond to:

[0072] The chassis cell CPA / GS115 expressing carboxypeptidase A localization fusion functional protein has the deposit number: CGMCC No.29610;

[0073] The chassis cell CPA / X33 expressing carboxypeptidase A localization fusion functional protein has the deposit number: CGMCC No.29609;

[0074] The chassis cell CPA / HCPB-GS115 co-expressing carboxypeptidase A and carboxypeptidase B localization fusion functional protein has the deposit number: CGMCC No.29607;

[0075] The chassis cell CPA / HCPB-X33 co-expressing carboxypeptidase A and carboxypeptidase B localization fusion functional protein has the deposit number: CGMCC No.29608;

[0076] The above chassis cell storage addresses are all: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the storage date is all: January 12, 2024, and the classification name is all: Komagataella phaffii.

[0077] (2) Induced expression and identification:

[0078] The above-mentioned chassis cells CPA / HCPB-GS115 and CPA / HCPB-X33 were placed in a 100 mL Erlenmeyer flask filled with 15 mL BMGY medium and cultured at 30°C and 220 rpm until OD600 was about 2 to 6 (16 to 18 h). Centrifuge at 1500 to 3000 g for 5 min at room temperature, collect the cells, resuspend the cells in BMMY medium to about OD600 of 2, place on a shaker at 30°C and 220 rpm and continue to grow for more than 36 h, and add 100% methanol to the culture medium every 24 h to a final concentration of 1.0% (v / v).

[0079] The bacterial liquid was collected and centrifuged at 12000g for 5 min, the bacteria were collected, and lysed with 8M urea. After the samples were prepared, Western Blot detection was performed using Anti-Carboxypeptidase A antibody (purchased from abcam), rabbit anti-CPB1 polyclonal antibody (purchased from Sangon Biotechnology (Shanghai) Co., Ltd.) and Anti-Myc antibody (purchased from Sangon Biotechnology (Shanghai) Co., Ltd.), ECL chemiluminescence reagent was used for color development, and the results were detected using a fully automatic chemiluminescence image analysis system (Tanon5200) to synthesize the image.

[0080] Test results such as Figure 2 As shown in the figure, it can be seen that lanes 1 to 4 are CPA / HCPB-GS115 bacterial lysis samples, and lanes 5 to 8 are CPA / HCPB-X33 bacterial lysis samples. The target bands that meet the expected size of the fusion protein appear, indicating that carboxypeptidase A is successfully expressed in the cell without affecting the expression of carboxypeptidase B. This shows that strains CPA / HCPB-GS115 and CPA / HCPB-X33 can express carboxypeptidase A and carboxypeptidase B simultaneously in the cell after methanol induction.

[0081] Example 3: Enzyme cleavage effect verification

[0082] The Kex2 restriction site P3 position was designed to verify the restriction effect of small molecule collagen sequences COL3LF (SEQ ID NO: 10) and COL3LV (SEQ ID NO: 11). The protein sequences of COL3LF and COL3LV were reverse translated according to the Pichia pastoris preferred codon to obtain DNA sequences, and Nanjing GenScript Technology Co., Ltd. was commissioned to synthesize the DNA sequences encoding COL3LF and COL3LV, which were cloned into the expression vector pPIC9K (Thermo Fisher Scientific (China) Co., Ltd.) between the α-factor secretion signal / cleavage site 1203 (search sequence is AAAGAAGAAGGGGTATCTCTCGAGAAAAGA (SEQ ID NO: 12)) and the Not I restriction site, respectively, and the expression frame was consistent with the α-factor secretion signal, and the recombinant plasmids pPIC9K-COL3LF and pPIC9K-COL3LV expressing COL3LF and COL3LV were obtained.

[0083] COL3LF (SEQ ID NO: 10):

[0084] EAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGLFKREA GKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGLFKR;

[0085] COL3LV (SEQ ID NO: 11):

[0086] EAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGLVKREA GKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGLVKR.

[0087] The monomer amino acid sequences of COL3LF and COL3LV are shown in SEQ ID NO: 13 and SEQ ID NO: 14:

[0088] COL3LF monomer (SEQ ID NO: 13):

[0089] EAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGLFK R;

[0090] COL3LV monomer (SEQ ID NO: 14):

[0091] EAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGLVK R.

[0092] 10 μg of the obtained recombinant plasmid was taken and digested with restriction endonuclease SacI (purchased from Dalian Takara Company) at 37°C to linearize it, and then the linearized plasmid was recovered using a PCR product purification kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.).

[0093] 10 μL of linearized plasmid was electroporated into Pichia pastoris CPA / HCPB-GS115 competent cells, and then 200 μL of the transformation product was spread on the MD plate, and then inverted and cultured in a 30°C incubator for 2-3 days until positive transformants appeared. After the positive transformants appeared, 2 mL of sterile double distilled water was added to the surface of the MD plate, and then the His+ transformants on the surface of the plate were gently scraped off with a sterile triangular spreader and transferred to a sterile centrifuge tube, and then diluted with sterile double distilled water to obtain a bacterial suspension. The bacterial suspension was diluted to 10 5The cells were spread on a YPD plate containing 0.5 mg / mL G418 and inverted in a 30°C incubator for 3 to 4 days until a single colony appeared, thereby obtaining Pichia pastoris engineered bacteria expressing COL3LF and COL3LV small molecule collagens, respectively.

[0094] The obtained Pichia yeast expressing the above COL3LF and COL3LV small molecule collagen was placed in a 100 mL Erlenmeyer flask containing 15 mL BMGY medium, and cultured at 30°C and 220 rpm until OD600 was about 2-6 (16-18 h). After the culture was completed, the cells were centrifuged at 1500-3000 g for 5 min at room temperature, and the cells were collected. The cells were resuspended in BMMY medium to make OD600 about 2, and placed on a shaker at 30°C and 220 rpm to continue growing for more than 36 h. 100% methanol was added to the culture medium every 24 h to a final concentration of 1.0%. After the growth was completed, the bacterial liquid sample was collected and placed in a 1.5 mL EP tube, centrifuged at 12000 g for 5 min at 4°C, and the expression supernatant was collected. The sample was boiled in a metal bath at 100°C for 10 min with 2× loading buffer (purchased from Shanghai Wansheng Haotian Biotechnology Co., Ltd.) for SDS-PAGE detection.

[0095] The electrophoresis results are as follows Figure 3 As shown in the figure, it can be seen that the target bands of the expected size appeared and the bands were single, indicating that the designed enzyme cleavage sites LFKREA and LVKREA can be recognized by Kex2 and efficiently cut to obtain monomeric small molecule collagen.

[0096] The target small molecule protein band on the SDS-PAGE gel was cut out and sent to Beijing Biotech Biotechnology Co., Ltd. for C-terminal sequence identification using Easy nanoLC 1200 / Q-Exactive. Figure 4 They are the secondary mass spectra of samples COL3LF and COL3LV, and the identification results are shown in Table 1.

[0097] Table 1. Target small molecule protein band identification results

[0098]

[0099]

[0100] Combining Table 1 and Figure 4It can be seen that after carboxypeptidase B removed the terminal dibasic amino acid KR of the monomer small molecule, carboxypeptidase A gradually removed the newly generated terminal amino acid residues. Since glycine has an inhibitory effect on the cleavage of carboxypeptidase A, no further resection was detected after the resection of LF and LV, indicating that carboxypeptidase A and carboxypeptidase B enzymes synergistically removed the restriction sites of non-target protein sequences added in the repeated tandem expression of small molecule collagen.

[0101] In summary, the present invention constructs a localized fusion functional protein, and constructs a chassis cell based on the localized fusion functional protein. The chassis cell can be specifically used for the repeated tandem expression of recombinant small molecule collagen, and can completely remove the double basic amino acids of the Kex2 enzyme cleavage recognition site connecting the tandem monomers and the specific amino acids added before and after the double basic amino acids for improving the Kex2 recognition and cleavage efficiency in the protein secretion expression pathway, so as to obtain a recombinant small molecule collagen that is 100% homologous to the natural human collagen sequence. In the process of designing the repeated tandem sequence, the Kex2 enzyme cleavage efficiency is guaranteed, and the introduced amino acids do not necessarily have to be the collagen's own amino acids, which greatly improves the design space and has good practicality.

[0102] The embodiments are preferred implementations of the present invention, but the present invention is not limited to the above-mentioned implementations. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essential content of the present invention belong to the protection scope of the present invention.

Claims

1. A localized fusion functional protein, characterized in that: The localization fusion functional protein includes carboxypeptidase A and a functional region having intracellular membrane localization or conversion and transportation functions between various cell organelles.

2. The localization fusion functional protein according to claim 1, characterized in that: The localization fusion functional protein also includes a connection sequence, which is used for connecting carboxypeptidase A with a functional region sequence having intracellular membrane localization or conversion and transport functions between organelles when fused and expressed.

3. The localization fusion functional protein according to claim 2, characterized in that: The connection sequence is a Linker sequence, as shown in SEQ ID NO.

5.

4. The localization fusion functional protein according to claim 1, characterized in that: The functional region sequence with intracellular membrane localization or conversion and transport between organelles is derived from Kex2 of Saccharomyces cerevisiae or Pichia pastoris, or other protein functional regions with similar functions such as Ste13 protease.

5. The localization fusion functional protein according to claim 4, characterized in that: The sequence of the functional region having the function of intracellular membrane localization or conversion and transport between organelles is shown in SEQ ID NO:

4.

6. The localization fusion functional protein according to any one of claims 1 to 5, characterized in that: The N-terminal of the localized fusion functional protein is fused with an α-factor secretion signal signal peptide sequence, and the C-terminal is fused with a myc tag.

7. The localization fusion functional protein according to any one of claims 1 to 6, characterized in that: The amino acid sequence of the localized fusion functional protein includes SEQ ID NO: 1 or an amino acid sequence that is 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to SEQ ID NO:

1.

8. A nucleic acid, characterized in that The nucleic acid encodes the localization fusion functional protein according to any one of claims 1 to 7.

9. The nucleic acid according to claim 8, characterized in that The nucleic acid includes the nucleotide sequence shown in SEQ ID NO: 2, or a degenerate sequence thereof.

10. A recombinant expression vector comprising the nucleic acid of claim 8 or 9.

11. The recombinant expression vector according to claim 10, characterized in that: The method for constructing the recombinant expression vector comprises: Cloning the nucleic acid encoding the localization fusion functional protein according to any one of claims 1 to 7 into plasmid 1 to obtain recombinant plasmid 1, and assembling the recombinant plasmid 1 with plasmid 2 to obtain recombinant plasmid 2; The recombinant plasmid 2, the DAS1 promoter and the AOX terminator are cloned into the plasmid 3 to obtain the recombinant expression vector.

12. The recombinant vector according to claim 11, characterized in that The plasmid 1 includes pUC57-BasI-Free; the plasmid 2 includes BB1_23; and the plasmid 3 includes BB3eH_14.

13. A chassis cell comprising the nucleic acid of claim 8 or 9, or comprising the recombinant vector of any one of claims 10 to 12, or expressing the localized fusion functional protein of any one of claims 1 to 7.

14. The chassis cell according to claim 13, characterized in that: The host bacteria of the chassis cells include Pichia pastoris, Saccharomyces cerevisiae, Hansenula, or bacteria expressing carboxypeptidase B localization fusion functional protein.

15. The chassis cell according to claim 14, characterized in that: The bacteria expressing carboxypeptidase B include GS115 / HCPB and X33 / HCPB.

16. The chassis cell according to claim 13, characterized in that: The chassis cells are deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, with the deposit numbers being CGMCC No.29610 and CGMCC No.29609.

17. The chassis cell according to any one of claims 13 to 14, characterized in that: The chassis cells also simultaneously express carboxypeptidase B localization fusion functional protein.

18. The chassis cell according to claim 17, characterized in that: The chassis cells are deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, with the deposit numbers being CGMCC No.29607 and CGMCC No.29608.

19. Use of the chassis cell according to any one of claims 13 to 18 in expressing recombinant small molecule collagen.

20. A method for obtaining recombinant small molecule collagen, characterized in that: The method comprises: The tandem repeat expression sequence of the recombinant small molecule collagen is connected to an expression vector and then transferred into the chassis cell according to any one of claims 13 to 18 to obtain a recombinant engineered bacterium containing or expressing the recombinant small molecule collagen, and the expression is induced by fermentation to obtain the recombinant small molecule collagen.

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