Codon optimization, expression, and characterization of an internalizing anti-ErbB2 single-chain antibody in Pichia pastoris.
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 Pichia 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- to 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 72 h 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.
Hu
Siyi
S
Laboratory of Molecular and Cellular Immunology, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, PR China.
Li
Liangwei
L
Qiao
Jingjuan
J
Guo
Yujie
Y
Cheng
Liansheng
L
Liu
Jing
J
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D003062:Codon / Q000096:biosynthesis* / Q000737:chemistry; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology*
D000911:Antibodies, Monoclonal; D003062:Codon; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D018719:Erb-b2 Receptor Tyrosine Kinases