Charge engineering of a protein domain to allow efficient ion-exchange recovery
2000 (English)In: Protein Engineering, ISSN 0269-2139, E-ISSN 1460-213X, Vol. 13, no 10, 703-709 p.Article in journal (Refereed) Published
We have created protein domains with extreme surface charge. These mutated domains allow for ion-exchange chromatography under conditions favourable for selective and efficient capture, using Escherichia coli as a host organism. The staphylococcal protein A-derived domain Z (Z(wt)) was used asa scaffold when constructing two mutants, Z(basic1) and Z(basic2), with high positive surface charge. Far-ultraviolet circular dichroism measurements showed that they have a secondary structure content comparable to the parental molecule 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 produced successfully as intracellular full-length products in E. coli and purified to homogeneity by ion-exchange chromatography. Further studies performed on Z(basic1) and Z(basic2) showed that they were able to bind to a cation 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 from streptococcal protein G, was also constructed. The gene product Z(basic2)-ABD could be purified using cation-exchange chromatography from a whole cell lysate to more than 90% purity.
Place, publisher, year, edition, pages
2000. Vol. 13, no 10, 703-709 p.
circular dichroism, ion-exchange chromatography, molecular modelling, pI, protein A, bacterial receptor domain, escherichia-coli k-12, fusion protein, binding domain, nucleic-acids, force-field, purification, dna, resolution, sequence
IdentifiersURN: urn:nbn:se:kth:diva-13275DOI: 10.1093/protein/13.10.703ISI: 000165995500006OAI: oai:DiVA.org:kth-13275DiVA: diva2:323036
QC 201006092010-06-092010-06-092012-06-13Bibliographically approved