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 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.
Hu
Shu-Hong
SH
Centre for Drug Design and Development and Special Research Centre for Functional and Applied Genomics, Institute for Molecular Bioscience, University of Queensland, Queensland Bioscience Precinct, Carmody Road, Brisbane, Qld 4072, Australia.
Gee
Christine L
CL
Latham
Catherine F
CF
Rowlinson
Scott W
SW
Rova
Ulrika
U
Jones
Alun
A
Halliday
Judy A
JA
Bryant
Nia J
NJ
James
David E
DE
Martin
Jennifer L
JL
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D015870:Gene Expression; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D015202:Protein Engineering; D050765:Qa-SNARE Proteins; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D008565:Membrane Proteins; D050765:Qa-SNARE Proteins; D011993:Recombinant Fusion Proteins; D006639:Histidine