Enhanced recombinant factor VII expression in Chinese hamster ovary cells by optimizing signal peptides and fed-batch medium.
Signal peptides play an important role in directing and efficiently transporting secretory proteins to their proper locations in the endoplasmic reticulum of mammalian cells. The aim of this study was to enhance the expression of recombinant coagulation factor VII (rFVII) in CHO cells by optimizing the signal peptides and type of fed-batch culture medium used. Five sub-clones (O2, I3, H3, G2 and M3) with different signal peptide were selected by western blot (WB) analysis and used for suspension culture. We compared rFVII expression levels of 5 sub-clones and found that the highest rFVII expression level was obtained with the IgK signal peptide instead of Ori, the native signal peptide of rFVII. The high protein expression of rFVII with signal peptide IgK was mirrored by a high transcription level during suspension culture. After analyzing culture and feed media, the combination of M4 and F4 media yielded the highest rFVII expression of 20 mg/L during a 10-day suspension culture. After analyzing cell density and cell cycle, CHO cells feeding by F4 had a similar percentage of cells in G0/G1 and a higher cell density compared to F2 and F3. This may be the reason for high rFVII expression in M4+F4. In summary, rFVII expression was successfully enhanced by optimizing the signal peptide and fed-batch medium used in CHO suspension culture. Our data may be used to improve the production of other therapeutic proteins in fed-batch culture.
a The Key Laboratory of Industrial Biotechnology , Ministry of Education, School of Biotechnology, Jiangnan University , Wuxi , China.
Lin
L
Peng
b Laboratory of Molecular Pharmacology , School of Pharmaceutical Sciences, Jiangnan University , Wuxi , China.
Xiao
X
Yu
b Laboratory of Molecular Pharmacology , School of Pharmaceutical Sciences, Jiangnan University , Wuxi , China.
Chengyuan
C
Li
b Laboratory of Molecular Pharmacology , School of Pharmaceutical Sciences, Jiangnan University , Wuxi , China.
Yanfei
Y
Cai
b Laboratory of Molecular Pharmacology , School of Pharmaceutical Sciences, Jiangnan University , Wuxi , China.
Yun
Y
Chen
c Jiangsu Institute of Hematology, the First Affiliated Hospital of Soochow University , Suzhou , China.
Yang
Y
He
d Cyrus Tang Hematology Center and Ministry of Education Engineering Center of Hematological Disease , Soochow University , Suzhou , China.
Jianfeng
J
Yang
b Laboratory of Molecular Pharmacology , School of Pharmaceutical Sciences, Jiangnan University , Wuxi , China.
Jian
J
Jin
a The Key Laboratory of Industrial Biotechnology , Ministry of Education, School of Biotechnology, Jiangnan University , Wuxi , China.
Huazhong
H
Li
D000818:Animals; D061249:Batch Cell Culture Techniques; D016466:CHO Cells; D002452:Cell Count; D002453:Cell Cycle / Q000235:genetics; D002470:Cell Survival; D003001:Cloning, Molecular / Q000379:methods*; D003412:Cricetulus; D003470:Culture Media / Q000737:chemistry; D005167:Factor VII / Q000096:biosynthesis / Q000235:genetics*; D005786:Gene Expression Regulation; D007136:Immunoglobulins / Q000235:genetics* / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D018741:Replication Origin; D014158:Transcription, Genetic
D003470:Culture Media; C014609:IgK; D007136:Immunoglobulins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D005167:Factor VII
CHO; SFM; cell cycle; rFVII; signal peptide