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Codon optimization, expression and characterization of an internalizing anti-ErbB2 single chain antibody in Pichia pastoris
Hu Siyi,Li Liangwei,Qiao Jingjuan ,Guo Yujie ,Cheng Liansheng ,Liu Jing
University of Science & Technology of China
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Funding: 教育部博士点基金（No.20020358048）
Opened online:13 March 2006
Accepted by: none
Citation: Hu Siyi,Li Liangwei,Qiao Jingjuan .Codon optimization, expression and characterization of an internalizing anti-ErbB2 single chain antibody in Pichia pastoris[OL]. [13 March 2006] http://en.paper.edu.cn/en_releasepaper/content/5672
 
 
Anti-ErbB2 antibodies are used as convenient tools in exploration of ErbB2 functional mechanisms and in treatment of ErbB2-overexpressing tumors. When we employed the yeast P. pastoris to express an anti-ErbB2 single chain antibody (scFv) derived from the tumor-inhibitory monoclonal antibody A21, the yield did not exceed 1-2 mg/L in shake flask cultures. As we considered that the poor codon usage bias may be one limiting factor leading to the inefficient translation and scFv production, we designed and synthesized the full-length scFv gene by choosing the P. pastoris preferred codons while keeping the G+C content at relatively low level. Codon optimization increased the scFv expression level 3-5 fold and up to 6-10 mg/L. Northern blotting further confirmed that the increase of scFv expression was mainly due to the enhancement of translation efficiency. Investigation of culture conditions revealed that the maximal cell growth and scFv expression were achieved at pH 6.5-7.0 with 2% casamino acids after 72h methanol induction. Secreted scFv was easily purified (>95% homogeneous product) from culture supernatants in one-step by using Ni2+ chelating affinity chromatography. The yield was approximately 10-15 mg/L. Functional studies showed that the A21 scFv could be internalized with high efficiency after binding to the ErbB2-overexpressing cells, suggesting this regent may prove especially useful for ErbB2-targeted immunotherapy.
Keywords:ErbB2; scFv; P. pastoris; Codon optimization; Internalization
 
 
 

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