Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria.

Michael-Paul Robinson,Na Ke,Corinna Tuckey,Thomas J Mansell,Julie Lobstein,Paul A Colussi,Paul D Riggs,Alana Szkodny,Cristen Peterson,Mehmet Berkmen,Christopher H Taron,Matthew P Delisa,Christopher J Noren

doi:10.1038/ncomms9072

Abstract

Current methods for producing immunoglobulin G (IgG) antibodies in engineered cells often require refolding steps or secretion across one or more biological membranes. Here, we describe a robust expression platform for biosynthesis of full-length IgG antibodies in the Escherichia coli cytoplasm. Synthetic heavy and light chains, both lacking canonical export signals, are expressed in specially engineered E. coli strains that permit formation of stable disulfide bonds within the cytoplasm. IgGs with clinically relevant antigen- and effector-binding activities are readily produced in the E. coli cytoplasm by grafting antigen-specific variable heavy and light domains into a cytoplasmically stable framework and remodelling the fragment crystallizable domain with amino-acid substitutions that promote binding to Fcγ receptors. The resulting cytoplasmic IgGs—named ‘cyclonals'—effectively bypass the potentially rate-limiting steps of membrane translocation and glycosylation.

Highlights

Current methods for producing immunoglobulin G (IgG) antibodies in engineered cells often require refolding steps or secretion across one or more biological membranes
Periplasmic expression is thought to be limited by the smaller volume and the lack of adenosine triphosphate (ATP)-dependent molecular chaperones in this compartment, as well as by the need for extensive optimization to efficiently secrete both the IgG heavy chain (HC) and light chain (LC) across the tightly sealed cytoplasmic membrane
No IgG activity above background was observed in WT E. coli cells expressing the anti-MBP cyclonal (Fig. 1b), consistent with the earlier observations that IgGs do not fold correctly in a normal reducing cytoplasm[24,25,26]

Summary

Introduction

Current methods for producing immunoglobulin G (IgG) antibodies in engineered cells often require refolding steps or secretion across one or more biological membranes. Periplasmic expression is thought to be limited by the smaller volume and the lack of adenosine triphosphate (ATP)-dependent molecular chaperones in this compartment, as well as by the need for extensive optimization to efficiently secrete both the IgG heavy chain (HC) and light chain (LC) across the tightly sealed cytoplasmic membrane To address these limitations, several groups have attempted to produce soluble IgGs in the cytoplasm of E. coli; none have been successful[24,25,26]. The end result is an entirely cytoplasmic system for efficient biosynthesis of immunologically and therapeutically relevant IgGs without the need for membrane translocation or glycosylation This platform provides a powerful complement to the existing antibody expression toolkit, but should open the door to a range of applications such as the rapid conversion of phage-displayed scFvs into full-length IgGs or animal-derived IgGs into humanized clones

Methods

Results

Conclusion