Expressing anti-HIV VRC01 antibody using the murine IgG1 secretion signal in Pichia pastoris

Abstract

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 materialThe online version of this article (doi:10.1186/s13568-017-0372-7) contains supplementary material, which is available to authorized users.

A streamlined cloning workflow minimising the time-to-strain pipeline for Pichia pastoris

“…Here, we separated the 5′ light (V L ) chain from the 3′ heavy (V H ) chain genes with the codon optimised T2A sequence from Thosea asigna virus, which was encoded in the primer sequences and was selected based on previous results showing a lower variance in processing efficiency 21 . The murine secretion signals previously used for expression 22 were amplified concurrent with both chains, and the backbone amplification moved upstream of the α-mating factor secretion signal to remove it from the final construct. The three parts of r_αA (3177 bp), VL-T2A (763 bp) and T2A-VH (1497 bp) were amplified and used to transform E. coli ; colony PCR results revealed a 25% assembly rate (n = 8) and a candidate was confirmed by sequencing.…”

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

“…Bacterial recombinant DNA manipulation was carried out in Escherichia coli strain NEB 5-α (New England Biolabs, Hertfordshire, UK). The VRC01 heavy and light chains were previously cloned into either pPICZα or pJANs-1 [ 7 ]. Vectors containing 10 different signal peptides were purchased from ATUM SM (Newark, California).…”

“…Genomic DNA was extracted using the DNeasy ® Plant Mini Prep Kit (Qiagen, Crawley, UK), quantified by Nanodrop™ (Thermo Fisher Scientific) and normalized to 0.5 ng μL −1 using distilled H 2 O. Quantitative PCR was run on genomic DNA using KICStart ® SYBR ® Green qPCR ReadyMix™ (Sigma Aldrich) in an Eppendorf Mastercycler ® ep realplex quantitative cycler (Eppendorf UK Ltd, Histon, UK). The known 1 copy clone α-1 was used as a reference strain and all strains were compared accordingly [ 7 ]. Data was analysed using the Pfaffl method, based on ΔΔ-Ct [ 37 , 38 ] and normalized to ACT1 as the housekeeping gene.…”

“…The antibody concentration reported by the gp140 ELISA method is lower than that of the total antibody method due to different binding affinities to the plate bound antigen. Yields of VRC01 for shake flasks were recorded previously [ 7 ].…”

“…We have previously reported on the production of the bNAb VRC01 using Pichia (syn. Komagataella) pastoris , with yields up to 3.05 mg L −1 in shake flasks [ 7 ]. Our research utilized the murine IgG1 signal peptide (GWSCIILFLVATATGVHSQ) for the first time in P. pastoris , which outperformed the more popular α-mating factor from Saccharomyces cerevisiae [ 8 , 9 ].…”