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Protein Expr Purif. 2018 Sep;149:43-50. doi: 10.1016/j.pep.2018.03.013. Epub 2018 Mar 27.

A systematic analysis of the expression of the anti-HIV VRC01 antibody in Pichia pastoris through signal peptide optimization.

Author information

1
Department of Life Sciences, Imperial College London, London, SW7 2AZ, UK; Centre for Synthetic Biology and Innovation, Imperial College London, SW7 2AZ, UK.
2
Department of Infectious Diseases, Imperial College London, London, W2 1PG, UK.
3
Department of Life Sciences, Imperial College London, London, SW7 2AZ, UK; Centre for Synthetic Biology and Innovation, Imperial College London, SW7 2AZ, UK. Electronic address: k.polizzi@imperial.ac.uk.

Abstract

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 mg L-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.

KEYWORDS:

2A sequence; Broadly neutralizing antibody; Pichia pastoris / Komagataella phaffi; Signal peptide; VRC01

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