DIRECTED EVOLUTION OF A TYPE-I ANTIFREEZE PROTEIN EXPRESSED IN ESCHERICHIA-COLI WITH SODIUM-CHLORIDE AS SELECTIVE PRESSURE AND ITS EFFECT ON ANTIFREEZE TOLERANCE

Both freezing tolerance and NaCl tolerance are improved when antifreez e proteins are expressed as fusion proteins with two domains of staphy lococcal protein A (SPA) in Escherichia coli. To characterize these pr operties further we created a randomly mutated expression library in E .coli, based on the winter flounder antifreeze protein HPLC-8 componen t gene. Low-fidelity PCR products of this gene were fused to the spa g ene encoding two domains of the SPA. The library was screened for enha nced NaCl tolerance and four clones were selected. The freezing tolera nce of each of the selected clones was enhanced to varying extents. DN A sequencing of the isolated mutants revealed that the amphiphilic pro perties of the native antifreeze protein were essentially conserved. F urthermore, by studying the primary sequence of the randomly mutated c lones, in comparison with the degree of freezing tolerance, we have id entified clues which help in understanding the relationship between sa lt and freezing tolerance.