High-level expression of a soluble and functional fibronectin type II domain from MMP-2 in the Escherichia coli cytoplasm for solution NMR studies.
We report a method for the expression in Escherichia coli of the isolated second type II fibronectin domain from MMP-2 (FNII-2). FNII-2 was expressed as a His(6)thioredoxin-tagged fusion protein in the thioredoxin reductase deficient E. coli strain BL21trxB(DE3), thus allowing disulfide-bond formation. When cultured at 37 degrees C, the expressed protein is located exclusively in the soluble fraction of the E. coli lysate. The fusion protein from the soluble fraction was purified and the His(6)thioredoxin-tag was cleaved by thrombin, resulting in a yield of approximately 40 mg/L. The recombinant FNII-2 was demonstrated to be functional by its ability to bind to gelatin-Sepharose, correct folding of the purified protein was confirmed by NMR spectroscopy. This approach may generally be applicable to all FNII domains and is a significant simplification relative to existing techniques involving refolding from inclusion bodies or expression in the eukaryotic host, Pichia pastoris.
Peisley
Alys A
AA
Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Vic. 3010, Australia.
Gooley
Paul R
PR
D002846:Chromatography, Affinity; D003593:Cytoplasm / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000166:cytology / Q000378:metabolism*; D005353:Fibronectins / Q000737:chemistry* / Q000145:classification; D015870:Gene Expression; D005822:Genetic Vectors; D006639:Histidine / Q000737:chemistry; D007553:Isotope Labeling; D020778:Matrix Metalloproteinase 2 / Q000737:chemistry* / Q000145:classification; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013879:Thioredoxins / Q000378:metabolism
D004220:Disulfides; D005353:Fibronectins; D011993:Recombinant Fusion Proteins; D006639:Histidine; D013879:Thioredoxins; D020778:Matrix Metalloproteinase 2