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Protein Engineering, Vol. 13, No. 10, 703-709, October 2000
© 2000 Oxford University Press

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

Torbjörn Gräslund1, Gunnel Lundin2, Mathias Uhlén1, Per-Åke Nygren1 and Sophia Hober1,3

1 Department of Biotechnology, Royal Institute of Technology (KTH), S-100 44 Stockholm and 2 Department of Genetics, Stockholm University, S-106 91 Stockholm, Sweden

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 (Zwt) was used as a scaffold when constructing two mutants, Zbasic1 and Zbasic2, with high positive surface charge. Far-ultraviolet circular dichroism measurements showed that they have a secondary structure content comparable to the parental molecule Zwt. Although melting temperatures (Tm) 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 Zbasic1 and Zbasic2 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 Zbasic2 and the acidic human serum albumin binding domain (ABD), derived from streptococcal protein G, was also constructed. The gene product Zbasic2–ABD could be purified using cation-exchange chromatography from a whole cell lysate to more than 90% purity.


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