期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 149, 期 -, 页码 43-50出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2018.03.013
关键词
Pichia pastoris / Komagataella phaffi; Broadly neutralizing antibody; VRC01; Signal peptide; 2A sequence
类别
资金
- Wellcome Trust
- EPSRC [EP/K038648/1]
- EPSRC [EP/K038648/1] Funding Source: UKRI
Pichia pastoris (Komagataella phaffi) has been used for recombinant protein production for over 30 years with over 5000 proteins reported to date. However, yields of antibody are generally low. We have evaluated the effect of secretion signal peptides on the production of a broadly neutralizing antibody (VRC01) to increase yield. Eleven different signal peptides, including the murine IgG1 signal peptide, were combinatorially evaluated for their effect on antibody titer. Strains using different combinations of signal peptides were identified that secreted approximately 2-7 fold higher levels of VRC01 than the previous best secretor, with the highest yield of 6.50 mgL(-1) in shake flask expression. Interestingly it was determined that the highest yields were achieved when the murine IgG1 signal peptide was fused to the light chain, with several different signal peptides leading to high yield when fused to the heavy chain. Finally, we have evaluated the effect of using a 2A signal peptide to create a bicistronic vector in the attempt to reduce burden and increase transformation efficiency, but found it to give reduced yields compared to using two independent vectors.
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