Construction Of Recombinant Protein Expression And Purification System For CHO Cells Based On SUMO-ULP1 Mutants

Objective:Construct CHO-S glutamine synthetase（GS）gene knockout cell lines by using CRISPR-Cas9 system.At the same time,the mutant SUMO tag was introduced into the GS complementary vector plasmid,and the Ulp1 protease mutant that recognized and cut the mutant SUMO was directed evolution,so as to establish a simple and rapid recombinant protein expression and purification system.Using this system to express and purify recombinant proteins with high value-added in the field of biomedicine,such as the organoid culture growth factor Noggin,facilitating the research and application of organoid culture.Method:1.CRISPR Lenti V2 plasmid or Cas9 RNP（Ribonucleoprotein,RNP）complex were used for genome editing in CHO-S cells respectively,and separately use puromycin screening or limited dilution method to screen for combined application with glutamine deficient culture medium.Eventually,CHO-S GS gene knockout cell lines were screened through Western Blot.2.Using the pc DNA3.1 eukaryotic cell expression vector as the plasmid skeleton,construction the eukaryotic expression vector carrying six histidine（His）tags of commercial SUMO mutants（SUMOstar）or self-designed SUMO mutants（named SUMOsm）-EGFP expression frame by molecular cloning technology.3.Transfer the pc DNA3.1-（6×His）-SUMOsm-EGFP and pc DNA3.1-（6×His）-SUMOstar-EGFP plasmids into HEK-293T cells.By comparing the expression of EGFP with two mutant SUMO tags in HEK-293T using fluorescence microscopy and Western Blot,the sensitivity of SUMO mutations to endogenous cell ULP1 was determined.4.According to the structural research results of SUMO-ULP1,a mutant ULP1 that can recognize and digest SUMOsm was designed.The mutant ULP1 was expressed by Escherichia coli DE3 star,and the mutant ULP1 protease was purified by nickel affinity chromatography.The SUMOsm-EGFP fusion protein expressed in HEK-293T cells was obtained by nickel metal affinity chromatography,and was used as the substrate for the ULP1 protease digesting substrate experiment in vitro.The efficiency of the ULP1 mutant digesting SUMOsm was verified by EGFP Western blot,and then obtain the SUMO mutant that is insensitive to endogenous protease but sensitive to the ULP1 mutant of directed evolution in vitro.5.Using molecular cloning technology for the construction of the expression vector p Opti Vec-GS/DHFR of eukaryotic cell expression vector with GS or dihydrofolate reductase（DHFR）as the screening marker,to express recombinant proteins in GS or DHFR deficient CHO cells.6.The Noggin reading frame was cloned into p Opti Vec-GS vector by molecular cloning technology,obtaining the p Opti Vec-GS-Noggin expression plasmid.The plasmid was transfected into CHO-S GS^-/-cells,screened and cultured in glutamine deficient medium,and the positive cell lines were attained.Expand the cultivation of monoclonal positive cell lines and collect cell supernatant to concentrate and enrich Noggin protein.Fianlly,using Western blot identify the expression of Noggin protein.Result:1.Successfully obtained the CHO-S cell line with GS knockout by using GS Cas9 plasmid and antibiotic screening,or GS Cas9 RNP complex and deficient culture medium screening.2.Two recombinant plasmids were successfully constructed for fusion expression of SUMO mutants and EGFP.They were transfected into HEK-293T cells and EGFP Western blot found that the self-designed SUMO mutants were more tolerant to endogenous ULP1 protease than the commercial SUMO mutants,making it easier to achieve the expression of SUMO fusion protein in eukaryotic cell.3.The mutant ULP1 protease was successfully expressed and purified in the Escherichia coli system,and it was verified by the enzyme digestion test that the mutant ULP1 protease could cut the self mutated SUMO-EGFP fusion protein,thus successfully evolving a pair of SUMO-ULP1mutants for eukaryotic cell expression.4.Successfully designed and constructed the recombinant plasmid p Opti Vec-GS/DHFR,Western blot results showed significant expression of the GS gene downstream of the IRES sequence,which confirmed the p Opti Vec-GS plasmid can be used for the expression of recombinant proteins in GS deficient CHO cells.5.CHO-S GS^-/-cells were transfected with the p Opti Vec-GS-Noggin expression plasmid,and screened by glutamine deficient medium,and then successfully obtained CHO stable cell lines expressing Noggin protein.Conclusion:Two delivery methods,GS Cas9 plasmid or Cas9 RNP complex,were used to transfect CHO-S cells,and the GS gene was knocked out through CRISPR-Cas9 system to obtain CHO-S GS^-/-nutrient deficient cell lines.The expression vector of eukaryotic cell with GS as the screening marker was successfully constructed to bring out the expression of Noggin protein in CHO GS^-/-cells.At the same time,SUMO-ULP1 mutant pair with independent intellectual property rights that can be used for eukaryotic cell expression has been successfully evolved,and new strategies have been explored for simplifying the expression and purification of recombinant protein in eukaryotic cell.