Expression And Identification Of The Codon-optimized Human PD-L1 Gene In Pichia Pastoris

Objective:To obtain soluble and immunogenic recombinant PD-L1 protein with Pichia pastoris,and to investigate the correlation between protein expression level and PDL1 gene copy number.To increase the protein expression level by increasing the copy number of the PD-L1 gene.Methods:The cDNA sequence of human PD-L1 published by GeneBank（PD-L1 transcript variation 1,NM₀14143.3）was selected,and codon optimization was carried out according to the preference of the Pichia pastoris.The codon-optimized human PD-L1 gene sequence was constructed into plasmid pPIC9K,and the recombinant plasmid pPIC9K-PDL1 was synthesized.The recombinant plasmid was linearized by restriction endonuclease Sac I-HF.40 μL competent yeast solution was respectively mixed with 10,5,and 1 μg linearized pPIC9K-PDL1 and electrocuted with a voltage of 1500 V,a capacitance of 25 F,and a resistance of 200 ω.After electrocuting,the bacteria solution was coated on MD plates（without histidine）and cultured at 30℃,3 d for positive transformant.Then,transformants grown on the MD plate were scraped off to make bacterial liquid.To screen multi-copy strains,the bacterial liquid was diluted and coated on YPD plates with G418 concentrations of 0.5,1.0,1.5,and 2.0 g/L,and cultured at 30℃ for 4 d.The colonies were selected from plates containing G418.PCR and 1%agarose gel electrophoresis were used to identify colonies.The positive colonies were sent for sequencing to confirm that the PDL1 gene was integrated into the yeast genome.The yeast genomic DNA was extracted as templates,the GAPDH gene of Pichia pastoris was used as a reference gene,and the target genes in selected yeast strains were determined by fluorescence quantitative PCR for relative quantification.Strains were divided into two groups:A（came from the plate with 1.0 g/L G418）and B（came from the plate with 1.5 g/L G418）,and the difference in relative copy number between the two groups was analyzed by unpaired t-test.The qualified strains were inoculated into the BMMY medium with 0.5%methanol and were induced in a 240 r/min shaker at 30℃.Supernatant samples of the medium were collected every 24 hours and methanol was supplemented.The culture medium supernatant was analyzed by SDSPAGE and Western blot.Results:（1）There was a positive correlation between the amount of plasmid added and the number of converters obtained after electric transformation.The number of converters between the 1 μg and the 10 μg group was statistically significant（Kruskal-Wallis H test,P=0.005）.（2）In this study,eight yeast strains were selected and identified to carry the PD-L1 gene.The strains in group A came from the YPD plate with 1.0 g/L G418,the relative copy numbers of the strains are 0.88,0.85,1.23,and 1.05.The strains in group B came from the YPD plate with 1.5 g/L G418,the relative copy numbers of the strains are 2.02,2.59,3.14,and 2.57.The relative copy number difference between the two groups was statistically significant（unpaired t-test,P=0.001）.The protein expression levels of A1～A4 and B1～B4 strains were 1.80,1.43,1.39,1,4.71,5.48,5.21,and 6.48,respectively,and the differences between the two groups were statistically significant（P<0.0001）.The average expression level of group B was 3.89 times more than that of group A.（3）The relative molecular weight of PD-L1 protein expressed in this study is about 44.7 kDa.The western blot results showed that the bands can specifically bind to the antiPD-L1 monoclonal antibody.（4）At the shaker level,the content of PD-L1 in the medium supernatant induced by 0.5%methanol for 72 h was the highest.Conclusion:（1）The engineering Pichia pastoris strains that stably expressed PD-L1 protein were successfully constructed,and the yield of PD-L1 could be maximized by harvesting culture medium supernatant at 72 h after being induced by 0.5%methanol.（2）The relative molecular weight of PD-L1 expressed by the strain was about 44.7 kDa,and it could specifically bind to the anti-PD-L1 monoclonal antibody.（3）Plates containing G418 can effectively screen multi-copy yeast strains and increase the yield of the target protein.The strain’s average protein expression increased 3.89 times after their copy number was lifted.