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


RESULTS
The 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 Valphabeta domain orientation was far superior to the Vbetaalpha domain orientation regarding monomeric yield of functionally folded molecules.


CONCLUSIONS
The 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.
Gunnarsen
Kristin S
KS
Centre for Immune Regulation, University of Oslo, Oslo, Norway. kristigu@imbv.uio.no
Lunde
Elin
E
Kristiansen
Per E
PE
Bogen
Bjarne
B
Sandlie
Inger
I
Løset
Geir Å
GÅ
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005822:Genetic Vectors; D008565:Membrane Proteins / Q000378:metabolism*; D018832:Molecular Chaperones / Q000378:metabolism*; D019696:Peptidylprolyl Isomerase / Q000378:metabolism*; D019897:Periplasm / Q000378:metabolism*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011948:Receptors, Antigen, T-Cell / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D018832:Molecular Chaperones; D011948:Receptors, Antigen, T-Cell; D019696:Peptidylprolyl Isomerase; C068254:FkpA protein, E coli