Pre-expression of a sulfhydryl oxidase significantly increases the yields of eukaryotic disulfide bond containing proteins expressed in the cytoplasm of E.coli

Kirsi Eh Salo,Eveliina Enlund,Van Dat Nguyen,Chi Zhang,Lloyd W Ruddock,Feras Hatahet

doi:10.1186/1475-2859-10-1

Abstract

BackgroundDisulfide bonds are one of the most common post-translational modifications found in proteins. The production of proteins that contain native disulfide bonds is challenging, especially on a large scale. Either the protein needs to be targeted to the endoplasmic reticulum in eukaryotes or to the prokaryotic periplasm. These compartments that are specialised for disulfide bond formation have an active catalyst for their formation, along with catalysts for isomerization to the native state. We have recently shown that it is possible to produce large amounts of prokaryotic disulfide bond containing proteins in the cytoplasm of wild-type bacteria such as E. coli by the introduction of catalysts for both of these processes.ResultsHere we show that the introduction of Erv1p, a sulfhydryl oxidase and a disulfide isomerase allows the efficient formation of natively folded eukaryotic proteins with multiple disulfide bonds in the cytoplasm of E. coli. The production of disulfide bonded proteins was also aided by the use of an appropriate fusion protein to keep the folding intermediates soluble and by choice of media. By combining the pre-expression of a sulfhydryl oxidase and a disulfide isomerase with these other factors, high level expression of even complex disulfide bonded eukaryotic proteins is possibleConclusionsOur results show that the production of eukaryotic proteins with multiple disulfide bonds in the cytoplasm of E. coli is possible. The required exogenous components can be put onto a single plasmid vector allowing facile transfer between different prokaryotic strains. These results open up new avenues for the use of E. coli as a microbial cell factory.

Highlights

Disulfide bonds are one of the most common post-translational modifications found in proteins
We show that the introduction of Erv1p along with a catalyst of disulfide bond isomerization into the cytoplasm of wild-type E. coli results in efficient production of eukaryotic disulfide bond containing proteins
Production of a tissue plasminogen activator fragment Our previous systems using Erv1p as a catalyst of disulfide bond formation in the cytoplasm of E. coli were based on screening for an increase in yield of active E. coli alkaline phosphatase (PhoA; 2 disulfides) and E. coli Phytase (AppA; 4 disulfides)

Summary

Introduction

Disulfide bonds are one of the most common post-translational modifications found in proteins. The production of proteins that contain native disulfide bonds is challenging, especially on a large scale. Either the protein needs to be targeted to the endoplasmic reticulum in eukaryotes or to the prokaryotic periplasm These compartments that are specialised for disulfide bond formation have an active catalyst for their formation, along with catalysts for isomerization to the native state. The formation of native disulfide bonds is often the rate limiting step of protein biogenesis [1,2,3]. In compartments where disulfide bond formation naturally occurs there are two distinct steps in Recently we showed that the introduction of a sulfhydryl oxidase, a natural catalyst of de novo disulfide bond formation, into the cytoplasm of E. coli allowed efficient production of disulfide bond containing prokaryotic proteins even without disruption of the reducing pathways [12]. In particular we used the sulfhydryl oxidase Erv1p from the inter-membrane mitochondrial space of S. cerevisiae

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