Periplasmic expression of soluble single chain T cell receptors is rescued by the chaperone FkpA

Inger Sandlie,Geir Å Løset,Per E Kristiansen,Bjarne Bogen,Kristin S Gunnarsen,Elin Lunde

doi:10.1186/1472-6750-10-8

Abstract

BackgroundEfficient expression systems exist for antibody (Ab) molecules, which allow for characterization of large numbers of individual Ab variants. In contrast, such expression systems have been lacking for soluble T cell receptors (TCRs). Attempts to generate bacterial systems have generally resulted in low yields and material which is prone to aggregation and proteolysis. Here we present an optimized periplasmic bacterial expression system for soluble single chain (sc) TCRs.ResultsThe effect of 1) over-expression of the periplasmic chaperon FkpA, 2) culture conditions and 3) molecular design was investigated. Elevated levels of FkpA allowed periplasmic soluble scTCR expression, presumably by preventing premature aggregation and inclusion body formation. Periplasmic expression enables disulphide bond formation, which is a prerequisite for the scTCR to reach its correct fold. It also enables quick and easy recovery of correctly folded protein without the need for time-consuming downstream processing. Expression without IPTG induction further improved the periplasmic expression yield, while addition of sucrose to the growth medium showed little effect. Shaker flask yield of mg levels of active purified material was obtained. The Vαβ domain orientation was far superior to the Vβα domain orientation regarding monomeric yield of functionally folded molecules.ConclusionThe general expression regime presented here allows for rapid production of soluble scTCRs and is applicable for 1) high yield recovery sufficient for biophysical characterization and 2) high throughput screening of such molecules following molecular engineering.

Highlights

Efficient expression systems exist for antibody (Ab) molecules, which allow for characterization of large numbers of individual Ab variants
The scTCRs are fused to an N -terminal pelB signal sequence that directs protein translocation across the inner membrane of E. coli through the SecB-dependent pathway to the oxidising environment of the periplasm
In pFKPEN, recombinant FkpA expression is controlled by its native promoter, while pFKPEI contains a dicistronic expression cassette where rFkpA gene transcription is controlled by the lac promoter

Summary

Introduction

Efficient expression systems exist for antibody (Ab) molecules, which allow for characterization of large numbers of individual Ab variants. Such expression systems have been lacking for soluble T cell receptors (TCRs). Recent re-evaluation and comparison of the existing pMHC/TCR crystal structures demonstrate conservation of specific TCR-MHC contacts in complexes bearing common V segments and MHC allotypes. This has for the first time made it possible to postulate a TCR-MHC recognition code [1,2,3,4,5]. Soluble TCRs are prone to aggregation and missfolding, and many of the difficulties encountered may probably be explained by the fact that TCRs, unlike Abs, have not evolved to be secreted, but are expressed as membranebound molecules that are intrinsically unstable when expressed as soluble molecules

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