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Recombinant expression of Munc18c in a baculovirus system and interaction with syntaxin4.
- Source :
-
Protein expression and purification [Protein Expr Purif] 2003 Oct; Vol. 31 (2), pp. 305-10. - Publication Year :
- 2003
-
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.
- Subjects :
- Animals
Gene Expression
Histidine genetics
Histidine metabolism
Membrane Proteins isolation & purification
Mice
Protein Engineering
Qa-SNARE Proteins
Rats
Recombinant Fusion Proteins genetics
Recombinant Fusion Proteins isolation & purification
Recombinant Fusion Proteins metabolism
Baculoviridae genetics
Membrane Proteins metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1046-5928
- Volume :
- 31
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- Protein expression and purification
- Publication Type :
- Academic Journal
- Accession number :
- 14550652
- Full Text :
- https://doi.org/10.1016/s1046-5928(03)00197-9