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.

Reliable titration of filamentous bacteriophages independent of pIII fusion moiety and genome size by using trypsin to restore wild-type pIII phenotype.

Phage display holds a key position in the use of combinatorial library approaches for the purpose of protein engineering and discovery. However, modifying the pIII protein of the phage can severely and negatively influence the infectiousness of the phage particle. This concern is particularly relevant when large pIII fusions in combination with multivalent display systems are in use. We here describe the use of trypsin to restore wild-type pIII phenotype as a small modification to the standard titration protocol. The results show that the trypsin treatment has a very large but heterogeneous effect on the phage infection efficiency, depending on the pIII fusion domain and the valence of display.