Charge engineering of a protein domain to allow efficient ion-exchange recovery

We have created protein domains with extreme surface charge. These mutated domains allow for ion-exchange chromatography under conditions favourable f or selective and efficient capture, using Escherichia coli as a host organi sm. The staphylococcal protein A-derived domain Z (Z(wt)) was used asa scaf fold when constructing two mutants, Z(basic1) and Z(basic2), with high posi tive surface charge. Far-ultraviolet circular dichroism measurements showed that they have a secondary structure content comparable to the parental mo lecule Z(wt). Although melting temperatures (T-m) of the engineered domains were lower than that of the wild-type Z domain, both mutants could be prod uced successfully as intracellular full-length products in E. coli and puri fied to homogeneity by ion-exchange chromatography. Further studies perform ed on Z(basic1) and Z(basic2) showed that they were able to bind to a catio n exchanger even at pH values in the 9 to 11 range. A gene fusion between Z (basic2) and the acidic human serum albumin binding domain (ABD), derived f rom streptococcal protein G, was also constructed. The gene product Z(basic 2)-ABD could be purified using cation-exchange chromatography from a whole cell lysate to more than 90% purity.