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

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 recom...

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Veröffentlicht in:Protein expression and purification 2003-10, Vol.31 (2), p.305-310
Hauptverfasser: Hu, Shu-Hong, Gee, Christine L, Latham, Catherine F, Rowlinson, Scott W, Rova, Ulrika, Jones, Alun, Halliday, Judy A, Bryant, Nia J, James, David E, Martin, Jennifer L
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Sprache:eng
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Zusammenfassung: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.
ISSN:1046-5928
1096-0279
1096-0279
DOI:10.1016/S1046-5928(03)00197-9