Purification of Active Chloroplast Sedoheptulose-1,7-Bisphosphatase Expressed in Escherichia coli

Authors: Dunford R.P.¹; Catley M.A.¹; Raines C.A.²; Lloyd J.C.²; Dyer T.A.¹

Source: Protein Expression and Purification, Volume 14, Number 1, October 1998 , pp. 139-145(7)

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Abstract:

Sedoheptulose-1,7-bisphosphatase (SBPase) is an enzyme unique to photosynthetic organisms and has a key role in regulating the photosynthetic Calvin cycle through which nearly all carbon enters the biosphere. This makes SBPase an appropriate target for intensive study. We have expressed wheat SBPase in Escherichia coli either with or without an N-terminal polyhistidine tag. The identity of the recombinant SBPases was confirmed by SDS–PAGE analysis and immunological detection with a specific antibody. Recombinant SBPase with a polyhistidine tag (His-SBPase) was obtained in soluble, active form and purified by one-step metal-chelate chromatography. Like the native enzyme, recombinant His-SBPase was specific for the substrate sedoheptulose-1,7-bisphosphate and required the presence of a reducing agent for activity. Polyclonal antibodies were raised against recombinant SBPase and were then used to determine relative levels of the enzyme in plant extracts. The availability of large amounts of active recombinant SBPase will also allow detailed structural studies by site-directed mutagenesis and X-ray crystallography. Copyright 1998 Academic Press.

Language: English

Document Type: Research article

Affiliations: 1: John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, United Kingdom 2: Biology Department, University of Essex, Colchester, CO4 3SQ, United Kingdom

Publication date: 1998-10-01