期刊
MOLECULAR IMMUNOLOGY
卷 40, 期 10, 页码 717-722出版社
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.molimm.2003.08.006
关键词
antibody expression; codon pair use
资金
- NCI NIH HHS [CA87990] Funding Source: Medline
- NIAID NIH HHS [AI39187, AI29470] Funding Source: Medline
- NATIONAL CANCER INSTITUTE [R01CA087990] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES [R37AI029470, R01AI029470, R01AI039187] Funding Source: NIH RePORTER
Here, we report that a significant increase in recombinant fusion antibody expression can be accomplished by adjusting the nucleotide sequence to conform to certain codon pairing rules. We investigated the expression of a protein in which a single chain Fv specific for HER2/neu with V-H and V-L joined by a flexible (GGGGS)(3) linker was linked to the C(H)3 of a human anti-rat transferrin receptor IgG3 heavy chain with the same flexible (GGGGS)(3) linker. In initial experiments we failed to achieve significant expression of this protein. However, when we made a single nucleotide change in each (GGGGS)(3) linker we were able to achieve expression The change of one nucleotide within each linker did not alter either the amino acid sequence or the frequency score of these codon triplets' usage in mammalian cells. Instead they removed two codon pairs predicted to be detrimental to expression. In a transient transfection assay we find that this change results in an over 30-fold increase in expression that is not the result of an increase in the level of accumulated mRNA. In addition, the changes made it possible to isolate stably transfected mammalian cell clones producing high levels of fusion protein, which had not been possible using the original gene. (C) 2003 Elsevier Ltd. All rights reserved.
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