Recombinant expression of Munc18c in a baculovirus system and interaction with syntaxin4
| Autor: | Shu-Hong Hu, Ulrika Rova, Christine L. Gee, Jennifer L. Martin, Judy Halliday, Alun Jones, David E. James, Nia J. Bryant, Catherine F. Latham, S. W. Rowlinson |
|---|---|
| Rok vydání: | 2003 |
| Předmět: |
Qa-SNARE Proteins
Vesicle docking Recombinant Fusion Proteins Gene Expression Membrane Proteins Sf9 Biology Protein Engineering Fusion protein law.invention Protein–protein interaction Rats Vesicular transport protein Mice Affinity chromatography Biochemistry law Protein purification Recombinant DNA Animals Histidine Baculoviridae Biotechnology |
| Zdroj: | Protein expression and purification. 31(2) |
| ISSN: | 1046-5928 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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