Chaperone-assisted thermostability engineering of a soluble T cell receptor using phage display

TY - JOUR

T1 - Chaperone-assisted thermostability engineering of a soluble T cell receptor using phage display

AU - Gunnarsen, Kristin S

AU - Kristinsson, Solveig G

AU - Justesen, Sune

AU - Frigstad, Terje

AU - Buus, Søren

AU - Bogen, Bjarne

AU - Sandlie, Inger

AU - Løset, Geir Åge

PY - 2013/1/29

Y1 - 2013/1/29

N2 - We here report a novel phage display selection strategy enabling fast and easy selection of thermostabilized proteins. The approach is illustrated with stabilization of an aggregation-prone soluble single chain T cell receptor (scTCR) characteristic of the murine MOPC315 myeloma model. Random mutation scTCR phage libraries were prepared in E. coli over-expressing the periplasmic chaperone FkpA, and such over-expression during library preparation proved crucial for successful downstream selection. The thermostabilized scTCR(mut) variants selected were produced in high yields and isolated as monomers. Thus, the purified scTCRs could be studied with regard to specificity and equilibrium binding kinetics to pMHC using surface plasmon resonance (SPR). The results demonstrate a difference in affinity for pMHCs that display germ line or tumor-specific peptides which explains the tumor-specific reactivity of the TCR. This FkpA-assisted thermostabilization strategy extends the utility of recombinant TCRs and furthermore, may be of general use for efficient evolution of proteins.

AB - We here report a novel phage display selection strategy enabling fast and easy selection of thermostabilized proteins. The approach is illustrated with stabilization of an aggregation-prone soluble single chain T cell receptor (scTCR) characteristic of the murine MOPC315 myeloma model. Random mutation scTCR phage libraries were prepared in E. coli over-expressing the periplasmic chaperone FkpA, and such over-expression during library preparation proved crucial for successful downstream selection. The thermostabilized scTCR(mut) variants selected were produced in high yields and isolated as monomers. Thus, the purified scTCRs could be studied with regard to specificity and equilibrium binding kinetics to pMHC using surface plasmon resonance (SPR). The results demonstrate a difference in affinity for pMHCs that display germ line or tumor-specific peptides which explains the tumor-specific reactivity of the TCR. This FkpA-assisted thermostabilization strategy extends the utility of recombinant TCRs and furthermore, may be of general use for efficient evolution of proteins.

KW - Animals

KW - Cell Line

KW - Cell Line, Tumor

KW - Escherichia coli Proteins

KW - Membrane Proteins

KW - Mice

KW - Molecular Chaperones

KW - Multiple Myeloma

KW - Peptide Library

KW - Peptidylprolyl Isomerase

KW - Protein Engineering

KW - Receptors, Antigen, T-Cell

U2 - 10.1038/srep01162

DO - 10.1038/srep01162

M3 - Journal article

C2 - 23362461

SN - 2045-2322

VL - 3

SP - 1162

JO - Scientific Reports

JF - Scientific Reports

M1 - 1162

ER -