Chaperone-assisted thermostability engineering of a soluble T cell receptor using phage display.
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.
Centre for Immune Regulation, University of Oslo, N-0316 Oslo, Norway.
Kristin S
KS
Gunnarsen
Solveig G
SG
Kristinsson
Sune
S
Justesen
Terje
T
Frigstad
Søren
S
Buus
Bjarne
B
Bogen
Inger
I
Sandlie
Geir Åge
GÅ
Løset
D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018832:Molecular Chaperones; D009101:Multiple Myeloma / Q000235:genetics / Q000378:metabolism*; D019151:Peptide Library; D019696:Peptidylprolyl Isomerase / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D018832:Molecular Chaperones; D019151:Peptide Library; D011948:Receptors, Antigen, T-Cell; D019696:Peptidylprolyl Isomerase; C068254:FkpA protein, E coli