Finding a stabilized sequence for reconstructed Icosahedra Lumazine Synthase by random mutagenesis and Fluorescence-Activated Cell Sorting

Lumazine Synthase forms pentamers, C5 in some organism and icodahedra, Ih in other. The aim of this project is to investigate which evolutionary steps could have led the pentamers to assembly into icosahedral capsids. Therefore prevoius work has been performed to reconstruct ancestral sequences for the pentameric and icosahedral forms of Lumazine Synthase. The icosahedral sequence had been expressed and shown to give insoluble expression. The work in this thesis has therefore been done to, firstly, try to purify and refold the protein from inclusion bodies and secondly, try to improve the sequence to give soluble expression. Expression of the protein resulted in the protein in inclusion bodies. The protein could be purified from the inclusion bodies by guanidine·HCl extraction and size exclusion chromatography. However it was not possible to refold the protein into native structure by dialysis to remove the denturant, guanidine·HCl. To improve the sequence to give stable expression, a stability assay system was used in combination with random mutagenesis of the sequence. The stability assay system consist of two reporter proteins, red fluorescent tagRFP and green fluorescent sf -GFP. Expression of the protein is monitored by red fluorescence by tag RFP fused to the protein and the stability of the expressed protein is monitored by sf -GFP expressed under the control of stress activated DnaK promoter. The random mutagen- esis was performed by error prone PCR to produce fragments spanning the sequence of Lumazine Synthase. The created fragments were then used as megaprimers to amplify the whole plasmid containing the protein with tagRFP and the stability assay system. (Less)