Histidine Patch Thioredoxins
MUTANT FORMS OF THIOREDOXIN WITH METAL CHELATING AFFINITY THAT PROVIDE FOR CONVENIENT PURIFICATIONS OF THIOREDOXIN FUSION PROTEINS (*)
- Zhijian Lu,
- Elizabeth A. DiBlasio-Smith,
- Kathleen L. Grant(§),
- Nicholas W. Warne,
- Edward R. LaVallie,
- Lisa A. Collins-Racie,
- Maximillian T. Follettie,
- Mark J. Williamson and
- John M. McCoy(¶)
- ¶ To whom correspondence should be addressed: Genetics Inst., Inc., 87 Cambridge Park Dr., Cambridge, MA 02140. Tel.: 617-498-8225; Fax: 617-498-8878; :jmccoy{at}genetics.com.
Abstract
A cluster of surface amino acid residues on Escherichia coli thioredoxin were systematically mutated in order to provide the molecule with an ability to chelate metal ions. The combined effect of two histidine mutants, E30H and Q62H, gave thioredoxin the capacity to bind to nickel ions immobilized on iminodiacetic acid- and nitrilotriacetic acid-Sepharose resins. Even though these two histidines were more than 30 residues apart in thioredoxin's primary sequence, they were found to satisfy the geometric constraints for metal ion coordination as a result of the thioredoxin tertiary fold. A third histidine mutation, S1H, provided additional metal ion chelation affinity, but the native histidine at position 6 of thioredoxin was found not to participate in binding. All of the histidine mutants exhibited decreased thermal stability as compared with wild-type thioredoxin; however, the introduction of an additional mutation, D26A, increased their melting temperatures beyond that of wild-type thioredoxin. The metal chelating abilities of these histidine mutants of thioredoxin were successfully utilized for convenient purifications of human interleukin-8 and −11 expressed in E. coli as soluble thioredoxin fusion proteins.
Footnotes
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↵* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- IMAC
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immobilized metal ion affinity chromatography
- IDA
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iminodiacetic acid
- NTA
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nitrilotriacetic acid
- TrxA
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thioredoxin
- IL-8
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interleukin-8
- IL-11
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interleukin-11
- PAGE
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polyacrylamide gel electrophoresis
- Tricine
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N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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↵2E. A. DiBlasio-Smith and J. M. McCoy, manuscript in preparation.
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- Received November 8, 1995.
- Revision received December 7, 1995.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
