不同信号肽对GLP-1-Fc融合蛋白表达的影响
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摘要
目的探讨不同信号肽对GLP-1-Fc融合蛋白表达的影响。方法对含7种不同信号肽(A～G)GLP-1-Fc融合蛋白的谷氨酰胺缺陷型中国仓鼠卵巢细胞(CHOK1SV GS-KO)进行流加培养,评估含各信号肽细胞的比生长率,并检测融合蛋白的表达量、反相纯度、电荷异质性组成、降解比例及糖型。同时对含最优信号肽GLP-1-Fc融合蛋白的细胞进行高密度发酵,并检测生物活性。结果含信号肽E的重组细胞比生长率略低,相应的融合蛋白表达量也略低,但反相纯度较高,酸性异构体比例较低,主峰含量最高,且降解比例较低,糖型G0F/G0F含量最高,整体质量较好,因此确定其为最佳信号肽。含信号肽E的细胞在1 L和7 L细胞反应器中发酵,RP-HPLC纯度分别高达89. 66%和89. 76%,生物活性与市售参比制剂一致。结论信号肽的筛选和优化需综合评估信号肽对融合蛋白表达量和产物关键质量属性的影响。
Objective To investigate the effects of various signal peptides on expression of GLP-1-Fc fusion protein.Methods CHOK1 SV GS-KO cells containing the genes of GLP-1-Fc fusion protein with seven different signal peptides were subjected to fed-batch culture and evaluated for specific growth rate. The expressed fusion proteins with various signal peptides were determined for titer,reverse purity,charge variants,degradation ratio and glycan. The GLP-1-Fc fusion protein with optimal signal peptide was subjected to high density fermentation,and determined for biological activity. Results The specific growth rate of recombinant CHOK1 SV GS-KO cells containing signal peptide E was slightly low. However,the fusion protein with the signal peptide E showed slightly low titer,high reverse purity,low proportion of acidic isomers,low degradation ratio,as well as the highest main peak content and G0F/G0F content,thus signal peptide E was defined as the optimal signal peptide. The RP-HPLC purity of GLP-1 Fc fusion protein with signal peptide E fermented in 1 L and 7 L bioreactors were 89. 66% and 89. 76% respectively,of which the biological activities were consistent with those of commercial reference. Conclusion During the screening and optimization of signal peptide,the effects of signal peptide on titer and critical quality attributes of fusion proteins should be balanced.
Objective To investigate the effects of various signal peptides on expression of GLP-1-Fc fusion protein.Methods CHOK1 SV GS-KO cells containing the genes of GLP-1-Fc fusion protein with seven different signal peptides were subjected to fed-batch culture and evaluated for specific growth rate. The expressed fusion proteins with various signal peptides were determined for titer,reverse purity,charge variants,degradation ratio and glycan. The GLP-1-Fc fusion protein with optimal signal peptide was subjected to high density fermentation,and determined for biological activity. Results The specific growth rate of recombinant CHOK1 SV GS-KO cells containing signal peptide E was slightly low. However,the fusion protein with the signal peptide E showed slightly low titer,high reverse purity,low proportion of acidic isomers,low degradation ratio,as well as the highest main peak content and G0F/G0F content,thus signal peptide E was defined as the optimal signal peptide. The RP-HPLC purity of GLP-1 Fc fusion protein with signal peptide E fermented in 1 L and 7 L bioreactors were 89. 66% and 89. 76% respectively,of which the biological activities were consistent with those of commercial reference. Conclusion During the screening and optimization of signal peptide,the effects of signal peptide on titer and critical quality attributes of fusion proteins should be balanced.
引文
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[2] ZHOU Y L,LIU P,GAN Y T,et al. Enhancing full-length antibody production by signal peptide engineering[J]. Microb Cell Fact,2016,15:47.
[3] WANG X L,LIU H H,YUAN W Q,et al. Efficient production of CYTL1 protein using mouse IgGκsignal peptide in the CHO cell expression system[J]. Acta Biochim Biophys Sin,2016,48(4):391-394.
[4] MORI A,HARA S,SUGAHARA T,et al. Signal peptide optimization tool for the secretion of recombinant protein from Saccharomyces cerevisiae[J]. J Biosci Bioeng,2015,120(5):518-525.
[5] YING H,LIU H C. Identification of an alternative signal peptide cleavage site of mouse monoclonal antibodies by mass spectrometry[J]. Immunol Lett,2007,111(1):66-68.
[6] KOTIA R B,RAGHANI A R. Analysis of monoclonal antibody product heterogeneity resulting from alternate cleavage sites of signal peptide[J]. Anal Biochem,2010,399(2):190-195.
[7] BARASH S,WANG W,SHI Y G. Human secretory signal peptide description by hidden Markov model and generation of a strong artificial signal peptide for secreted protein expression[J]. Biochem Biophys Res Commun,2002,294(4):835-842.
[8] KOBER L,ZEHE C,BODE J. Optimized signal peptides for the development of high expressing CHO cell lines[J]. Biotechnol Bioeng,2013,110(4):1164-1173.
[9] HARYADI R,HO S,KOK Y J,et al. Optimization of heavy chain and light chain signal peptides for high level expression of therapeutic antibodies in CHO cells[J]. PLoS One,2015,10(2):e0116878. doi:10. 1371/journal. pone. 0116878.
[10] WEN B,DENG Y,GUAN J,et al. Signal peptide replacements enhance expression and secretion of hepatitis C virus envelope glycoproteins[J]. Acta Biochim Biophys Sin,2011,43(2):96-102.
[11] PENG L,YU X,LI C,et al. Enhanced recombinant factor VII expression in Chinese hamster ovary cells by optimizing signal peptides and fed-batch medium[J]. Bioengineered,2016,7(3):189-197.
[12] LIN J,NEO SH,HO S C L,et al. Impact of signal peptides on furin-2A mediated monoclonal antibody secretion in CHO cells[J]. Biotechnol J,2017,12(9):1-40.
[13] RAMEZANI A,MAHMOUDI MAYMAND E,YAZDANPANAH-SAMANI M,et al. Improving Pertuzumab production by gene optimization and proper signal peptide selection[J]. Protein Expr Purif,2017,135(1):24-32.
[14] GONG Y,CHEN C,LI Y D,et al. Effect of various signal peptides on expression of recombinant serogroup B Neisseria meningitidis factor H-binding protein[J]. Chin J Biologicals,2017,30(2):141-145,149.(in Chinese)宫悦,陈晨,李亚东,等.不同信号肽对重组B群脑膜炎奈瑟菌H因子结合蛋白表达的影响[J].中国生物制品学杂志,2017,30(2):141-145,149.