Expressing anti-HIV VRC01 antibody using the murine IgG1 secretion signal in Pichia pastoris.
The use of the recombinant expression platform Pichia pastoris to produce pharmaceutically important proteins has been investigated over the past 30 years. Compared to mammalian cultures, expression in P. pastoris is cheaper and faster, potentially leading to decreased costs and process development times. Product yields depend on a number of factors including the secretion signal chosen for expression, which can influence the host cell response to recombinant protein production. VRC01, a broadly neutralising anti-HIV antibody, was expressed in P. pastoris, using the methanol inducible AOX1 promoter for both the heavy and light chains. Titre reached up to 3.05 μg mL-1in small scale expression. VRC01 was expressed using both the α-mating factor signal peptide from Saccharomyces cerevisiae and the murine IgG1 signal peptide. Surprisingly, using the murine IgG1 signal peptide resulted in higher yield of antibody capable of binding gp140 antigen. Furthermore, we evaluated levels of secretory stress compared to the untransformed wild-type strain and show a reduced level of secretory stress in the murine IgG1 signal peptide strains versus those containing the α-MF signal peptide. As bottlenecks in the secretory pathway are often the limiting factor in protein secretion, reduced levels of secretory stress and the higher yield of functional antibody suggest the murine IgG1 signal peptide may lead to better protein folding and secretion. This work indicates the possibilities for utilising the murine IgG1 signal peptide for a range of antibodies, resulting in high yields and reduced cellular stress.
Department of Life Sciences, Imperial College London, London, SW7 2AZ, UK.|Centre for Synthetic Biology and Innovation, Imperial College London, London, SW7 2AZ, UK.
Rochelle
R
Aw
Department of Infectious Diseases, Imperial College London, London, W2 1PG, UK.
Paul F
PF
McKay
Department of Infectious Diseases, Imperial College London, London, W2 1PG, UK.
Robin J
RJ
Shattock
Department of Life Sciences, Imperial College London, London, SW7 2AZ, UK. k.polizzi@imperial.ac.uk.|Centre for Synthetic Biology and Innovation, Imperial College London, London, SW7 2AZ, UK. k.polizzi@imperial.ac.uk.
Karen M
KM
Polizzi
Alpha-mating factor signal peptide; Broadly neutralising antibody; Murine IgG1 signal peptide; Pichia pastoris/Komagataella phaffi; VRC01