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Recombinant expression of Munc18c in a baculovirus system and interaction with syntaxin4

Abstract

Two protein families that are critical for vesicle transport are the Syntaxin and Munc18/Sec1 families of proteins. These two molecules form a high affinity complex and play an essential role in vesicle docking and fusion. Munc18c was expressed as an N-terminally His-tagged fusion protein from recombinant baculovirus in Sf9 insect cells. His-tagged Munc18c was purified to homogeneity using both cobalt-chelating affinity chromatography and gel filtration chromatography. With this simple two-step protocol, 3.5 mg of purified Munc18c was obtained from a 1L culture. Further, the N-terminal His-tag could be removed by thrombin cleavage while the tagged protein was bound to metal affinity resin. Recombinant Munc18c produced in this way is functional, in that it forms a stable complex with the SNARE interacting partner, syntaxin4. Thus we have developed a method for producing and purifying large amounts of functional Munc18c--both tagged and detagged--from a baculovirus expression system. We have also developed a method to purify the Munc18c:syntaxin4 complex. These methods will be employed for future functional and structural studies.

Type Journal
ISBN 1046-5928 (Print)
Authors Hu, S. H.;Gee, C. L.;Latham, C. F.;Rowlinson, S. W.;Rova, U.;Jones, A.;Halliday, J. A.;Bryant, N. J.;James, D. E.;Martin, J. L. :
Publisher Name Protein Expr Purif
Published Date 2003-01-01 00:00:00
Published Volume 31
Published Issue 2
Published Pages 305-10
URL http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=14550652
Status Published In-print