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Charge engineering of a protein domain to allow efficient ion-exchange recovery
-
- Gräslund, Torbjörn (författare)
- KTH,Biokemi och biokemisk teknologi
- Lundin, Gunnel (författare)
-
- Uhlén, Mathias (författare)
- KTH,Biokemi och biokemisk teknologi
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- (creator_code:org_t)
- 2000-10
- 2000
- Engelska.
- Ingår i: Protein Engineering. - : Oxford University Press (OUP). - 0269-2139 .- 1460-213X. ; 13:10, s. 703-709
- Tidskriftsartikel (refereegranskat)
Abstract
Ämnesord
Stäng
- 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.
Ämnesord
- NATURVETENSKAP -- Biologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences (hsv//eng)
Nyckelord
- 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
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- Hober, Sophia
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