Protein Stability, Folding and Design: Stabilization of scFv Protein through its reconstitution using a split GFB biosensor in vivo

Abstract: General rules for rational design as well as prediction of tertiary structure and functionality of a protein can be described by investigating the interactions and the role of particular amino acids in protein structure. Mutagenesis has been used commonly to generate stable variants, with an ultimate goal to unravel the rules of protein stability and folding. Besides, reconstitution of dissected proteins has been used as well as an approach to find variants of particular proteins with increased affinity which could lead ultimately to enhancement of stability. In this project a random library of a hapten specific scFv, Anti Fluoroscien IsoThioCyanate (scFv) dissected into the fragments 1-124 (Heavy chain) and 125-246 (Light chain) was interrogated in order to find variants with improved affinity to be tested in further studies for stability enhancement of the corresponding intact protein variants. The split GFP system, a genetically codified biosensor, was used as a method to detect in vivo reconstitution of scFv (Heavy chain 1-124 + Light chain 125-246). Firstly, reconstitution of a single chain antibody (scFv) fragment 1-124 (Heavy chain) and 125-246 (Light chain) was detected. Secondly, a random library of the Light chain fragment 125-246 cloned into the GFP system was screened to find variants with higher fluorescence intensity than WT Light chain. An increase in fluorescence is suggested to arise from increased affinity which in turn could be used to select for stabilized intact variants. However we failed to detect green fluorescence. This may be due to problems in the expression of one of the partners (heavy chain-CGFP) or steric constraints and hence we were not able to screen any high affinity mutants. Various suggestions for improving the expression of the protein or relieving steric constraints are discussed here. If these problems are solved, libraries will be screened for the possible stabilizing role of the found substitutions. This can be discussed in terms of establishment of favorable hydrophobic interactions, stabilization of secondary structure and indirectly destabilization of the unfolded structure. The insight into the interactions and roles played by specific amino acids can be used to understand protein design of other proteins.