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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...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Springer Berlin Heidelberg
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5366992/ https://www.ncbi.nlm.nih.gov/pubmed/28342171 http://dx.doi.org/10.1186/s13568-017-0372-7 |
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author | Aw, Rochelle McKay, Paul F. Shattock, Robin J. Polizzi, Karen M. |
author_facet | Aw, Rochelle McKay, Paul F. Shattock, Robin J. Polizzi, Karen M. |
author_sort | Aw, Rochelle |
collection | PubMed |
description | 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(−1) in 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13568-017-0372-7) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-5366992 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Springer Berlin Heidelberg |
record_format | MEDLINE/PubMed |
spelling | pubmed-53669922017-04-12 Expressing anti-HIV VRC01 antibody using the murine IgG1 secretion signal in Pichia pastoris Aw, Rochelle McKay, Paul F. Shattock, Robin J. Polizzi, Karen M. AMB Express Original Article 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(−1) in 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13568-017-0372-7) contains supplementary material, which is available to authorized users. Springer Berlin Heidelberg 2017-03-24 /pmc/articles/PMC5366992/ /pubmed/28342171 http://dx.doi.org/10.1186/s13568-017-0372-7 Text en © The Author(s) 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. |
spellingShingle | Original Article Aw, Rochelle McKay, Paul F. Shattock, Robin J. Polizzi, Karen M. Expressing anti-HIV VRC01 antibody using the murine IgG1 secretion signal in Pichia pastoris |
title | Expressing anti-HIV VRC01 antibody using the murine IgG1 secretion signal in Pichia pastoris |
title_full | Expressing anti-HIV VRC01 antibody using the murine IgG1 secretion signal in Pichia pastoris |
title_fullStr | Expressing anti-HIV VRC01 antibody using the murine IgG1 secretion signal in Pichia pastoris |
title_full_unstemmed | Expressing anti-HIV VRC01 antibody using the murine IgG1 secretion signal in Pichia pastoris |
title_short | Expressing anti-HIV VRC01 antibody using the murine IgG1 secretion signal in Pichia pastoris |
title_sort | expressing anti-hiv vrc01 antibody using the murine igg1 secretion signal in pichia pastoris |
topic | Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5366992/ https://www.ncbi.nlm.nih.gov/pubmed/28342171 http://dx.doi.org/10.1186/s13568-017-0372-7 |
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