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

QC 20100609
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.

Lundin, Gunnel (författare)
Uhlén, MathiasKTH,Biokemi och biokemisk teknologi(Swepub:kth)u1dulvmw (författare)
Nygren, Per-ÅkeKTH,Biokemi och biokemisk teknologi(Swepub:kth)u1zhverl (författare)
Hober, SophiaKTH,Biokemi och biokemisk teknologi(Swepub:kth)u11qqzc1 (författare)
KTHBiokemi och biokemisk teknologi (creator_code:org_t)

Ingår i:Protein Engineering: Oxford University Press (OUP)13:10, s. 703-7090269-21391460-213X
Ingår i:Protein Engineering, Design and Selection: Oxford University Press (OUP)13:10, s. 703-7091741-01341741-0126

Av författaren/redakt...: Gräslund, Torbjö ...; Lundin, Gunnel; Uhlén, Mathias; Nygren, Per-Åke; Hober, Sophia

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