Stability of Functionally Active Fusion Proteins During Their Biosynthesis and Isolation from Expressing Bacterial Cells

Publisher Summary This chapter discusses the stability of functionally active fusion proteins during their biosynthesis and isolation from expressing bacterial cells. Several genetic constructs were created for the in vivo expression in E.coli cells of the following bimodular fusion proteins: glutation-S-transferase-sarcotoxin (GST-Sarc), sarcotoxin-obelin (Sarc-Ob), dihydrofolate reductase-obelin (DHFR-Ob), green fluorescence protein-obelin (GFP-Ob), barstar-obelin (Star-Ob). These proteins were used to demonstrate the effect of spacers and their nearest surrounding on the stability and functionality of individual modules in the fusion proteins. Treatment of the isolated GST-Sarc and GST with thrombin results in complete cleavage of the fusion protein with the formation of GST and, apparently, in complete degradation of sarcotoxin. The obtained data show that endogenous proteases present in the cell lysate primarily cleave the polypeptide chain at the region connecting the functional parts of the fusion protein to the parts without a rigid structure, resulting from the character of the amino acid sequence (impossibility of folding).