doi:10.1016/j.pep.2006.12.005
Copyright © 2006 Elsevier Inc. All rights reserved.
High-level expression of a soluble and functional fibronectin type II domain from MMP-2 in the Escherichia coli cytoplasm for solution NMR studies
References and further reading may be available for this article. To view references and further reading you must
purchase this article.
Alys A. Peisleya, b and Paul R. Gooleya, 
, 
aDepartment of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Vic. 3010, Australia
bDepartment of Pathology, Stanford School of Medicine, 300 Pasteur Drive, Stanford, CA 94305, USA
Received 23 October 2006;
revised 30 November 2006.
Available online 13 December 2006.
Abstract
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 His6thioredoxin-tagged fusion protein in the thioredoxin reductase deficient E. coli strain BL21trxB(DE3), thus allowing disulfide-bond formation. When cultured at 37 °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 His6thioredoxin-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.
Keywords: Matrix metalloprotease; MMP-2; Fibronectin type II domain
Fig. 1. Expression of FNII domain constructs in E. coli cytoplasm detected by Coomassie stained reduced SDS–PAGE fractionated proteins. (a) His6-tagged FNII-23, (b) His6-tagged FNII-123, and (c) His6-tagged FNII-2 and FNII-3 produced in BL21(DE3). Single colonies of transformed cells were grown as described in Materials and methods. Recombinant protein expression was induced with 1 mM IPTG and cells were harvested after 2 h of induction at 37 °C. Total cellular protein fraction was prepared by resuspending the bacterial pellet in 1/10 culture volume with PBS followed by addition of an equal volume of 4× SDS–PAGE sample buffer. Samples were sonicated briefly then boiled for 3 min prior to separation by SDS–PAGE. No expression of the single domain constructs FNII-2 and FNII-3 was detected.
Fig. 2. Thioredoxin tagged FNII-2 and FNII-3 expression from BL21trxB(DE3). Soluble and insoluble cytoplasmic fractions were prepared by cell lysis through enzymatic treatment with lysozyme and subsequent sonication followed by centrifugation at 16,000g for 10 min. Pellets were resuspended in 4× reducing SDS–PAGE sample buffer. Soluble and insoluble fractions were separated over 15% acrylamide reduced denaturing SDS–PAGE.
Fig. 3. Metal chelate affinity purification of His6Trx-tagged MMP-2-FNII-2 produced from BL21trxB(DE3). His6Trx-tagged MMP-2-FNII-2 was purified on Co-IDA agarose as described in Materials and methods. Steps in the purification were analyzed by 15% acrylamide SDS–PAGE. Lane 1, insoluble fraction; lane 2, soluble fraction; lane 3, unbound fraction; lane 4, wash fraction; lane 5, eluted fraction containing relatively pure His6Trx-MMP-2-FNII-2; lane 6, molecular weight marker.
Fig. 4. Gelatin-Sepharose 4B affinity chromatography of FNII-2. Thrombin cleaved MMP-2-FNII-2 was applied to a gelatin-Sepharose column equilibrated in 100 mM Tris–HCl, pH 7.4, 150 mM NaCl. Column was washed with one column volume of the same buffer and bound MMP-2-FNII-2 was then eluted with a 0–8 M urea gradient using an Econo gradient pump (BIORAD). Inset: final sample of purified MMP-2-FNII-2.
Fig. 5. 2D 1H,15N HSQC spectrum of 2 mM uniformly 15N-labeled FNII-2 in 20 mM sodium phosphate, pH 6.8 at 25 °C. Spectrum was acquired at 500 MHz with 1024 complex points in the 1H dimension and 256 complex points in the 15N dimension with spectral widths of 7100 and 1450 Hz in 1H and 15N dimensions, respectively. Resonance assignments were made to all peaks observed in the spectrum. Residue Q298 has been folded and has an actual chemical shift of 11.6 in 1H and 133.5 in 15N.
Table 1.
Steps in the purification of FNII-2a
a Protein concentrations were determined with the Bradford assay using BSA as a standard.
b 4.5–5.0 g of wet cells were obtained per liter of 2YT shaker flask or 250 mL M9 fermentation culture.
c Determined for Hisf6Trx-FNII-2 fusion protein.
Corresponding author. Fax: +61 3 9348 1421.
Abstract
Purchase PDF (394 K)
E-mail Article
Add to my Quick Links
Cited By in Scopus (0)
Purchase PDF (520 K)
