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 chromatographyunder conditions favourable for selective and efficient capture,using Escherichia coli as a host organism. The staphylococcalprotein A-derived domain Z (Z_wt) was used as a scaffold whenconstructing two mutants, Z_basic1 and Z_basic2, with high positivesurface charge. Far-ultraviolet circular dichroism measurementsshowed that they have a secondary structure content comparableto the parental molecule Z_wt. Although melting temperatures(T_m) of the engineered domains were lower than that of the wild-typeZ domain, both mutants could be produced successfully as intracellularfull-length products in E.coli and purified to homogeneity byion-exchange chromatography. Further studies performed on Z_basic1and Z_basic2 showed that they were able to bind to a cation exchangereven at pH values in the 9 to 11 range. A gene fusion betweenZ_basic2 and the acidic human serum albumin binding domain (ABD),derived from streptococcal protein G, was also constructed.The gene product Z_basic2–ABD could be purified using cation-exchangechromatography from a whole cell lysate to more than 90% purity.