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

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. and Martin, J. L. (2003) Recombinant expression of Munc18c in a baculovirus system and interaction with syntaxin4. Protein Expression And Purification, 31 2: 305-310. doi:10.1016/S1046-5928(03)00197-9

Author 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.
Title Recombinant expression of Munc18c in a baculovirus system and interaction with syntaxin4
Journal name Protein Expression And Purification   Check publisher's open access policy
ISSN 1046-5928
Publication date 2003
Sub-type Article (original research)
DOI 10.1016/S1046-5928(03)00197-9
Volume 31
Issue 2
Start page 305
End page 310
Total pages 6
Editor R. R. Burgess
Place of publication San Diego
Publisher Academic Press
Collection year 2003
Language eng
Subject C1
060107 Enzymes
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 1 L 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. Crown copyright (C) 2003 Published by Elsevier Inc. All rights reserved.
Keyword Biochemical Research Methods
Biochemistry & Molecular Biology
Biotechnology & Applied Microbiology
Snare Proteins
Protein Expression
Protein Purification
Protein Complex Formation
Protein-protein Interactions
Q-Index Code C1

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Created: Wed, 15 Aug 2007, 01:55:22 EST