Different lengths of linkers and expression host cell lines were tested in order to improve the yield of the MBP-S1R fusion protein. First attempts to express and purify MBP-SR1 replicate published results of our collaborator Professor A. Ruoho, who reported ~0.3 mg of protein/liter of cell culture. By decreasing the length of the amino acid linker, we increased the expression by a factor of 10 with the best-performing 3 alanine (AAA) and by a factor of 12 with 4 alanine (AAAA) linker construct.
Sigma-1 receptor (S1R) is a 223 amino acid eukaryotic membrane protein that is now recognized as chaperone and inter-organelle signaling modulator . Sigma-1 receptor is predicted to have two transmembrane domains and is categorized as a transmembrane protein.
The guinea pig S1R (gpS1R) was recently expressed as a properly folded and active protein in a bacterial host strain . In collaboration with Professor Ruoho’s team at the UW-Madison, we have expressed and purified gpS1R for functional studies and structure determination. To stabilize the protein, it is expressed and purified with maltose binding protein (MBP) that contains a periplasmic export sequence fused to the N-terminus of gpS1R. The expressed fusion protein (MBP-gpS1R) is anchored in the E. coli cytoplasmic membrane. We have designed a series of vectors with short linkers consisting of 0 to 5 alanine residues between the N-terminal MBP and the gpS1R receptor (Figure 1). All of these constructs were expressed in a series of E. coli expression host strains and purified at large scale (cell paste obtained from 4 L growth). To facilitate X-ray crystallography studies, MBP-gpS1R fusion is expressed as selenomethionyl-labeled protein, using modified defined medium and IPTG induction .
Our current purification procedure consists of : (1) extraction from membrane fraction by application of detergents; (2) purification by affinity chromatography (amylose resin); (3) gel filtration and subsequent concentration using centrifugation concentrators.
We are able to obtain up to 3.2 mg of purified MBP-gpS1R protein per liter of cell culture, with the final yield depending on the individual constructs linker (Figure 2). The MBP-gpS1R fusion protein retains ligand binding activity typical of native S1R. After initial optimization of MBP-S1R expression and purification, the best performing 3- (Figure 3, panel A) and 4-alanine linker (Figure 3, panel B) constructs were screened for highest yield of active protein in 5 different E. coli expression strains: B834-pRARE2, BL21(DE3), BL21(DE3)-RILP, C43(DE3)-pRARE2 and C43(DE3)-RILP. The C43 strains are recommended for expression of difficult proteins (including membrane proteins) but in case of MBP-S1R fusion protein the most successful expression host is the B834-pRARE2 strain. This has been consistent with all of the tested constructs. Panel B includes data for the best 3 alanine construct expression yield and activity level (orange bar) as comparison since ligand binding activity is calculated in different units.
Authors: Gromek KA, Wrobel RL, Frederick RO, Beebe ET, Aceti DJ, Misenheimer TM, Ruoho AE, Fox BG
Figure 1. The linker variations for the MBP-gpS1R fusion protein. The original construct from TMPC collaborator contains a linker with TEV protease cleavage site.
Figure 2. Screen of MBP-S1R construct yields after expression and purification. The yields after first chromatographic step (purification on amylose column) for all tested linkers and 2 different expression hosts. The best yields were obtained for the 3 and 4 alanine linkers contructs which were further tested to improve yields of active protein.
Figure 3. Optimization of MBP-S1R expression and purification. The best performing 3- (A) and 4-alanine linker (B) constructs were screened for highest yield of active protein in 5 different E. coli expression strains: B834-pRARE2, BL21(DE3), BL21(DE3)-RILP, C43(DE3)-pRARE2, and C43(DE3)-RILP. The C43 strains are recommended for expression of difficult proteins (including membrane proteins) but in case of MBP-S1R fusion protein the most successful expression host is the B834-pRARE2 strain. This has been consistent with all of the tested constructs. Panel B includes data for the best 3 alanine construct expression yield and activity level (orange bar) as comparison since ligand binding activity is calculated in different units.
Su TP, Hayashi T, Maurice T, Buch S, Ruoho AE. (2010) The sigma-1 receptor chaperone as an inter-organelle signaling modulator. Trends Pharmacol Sci 31 (12), 557-66. PubMed ID:20869780 | PMC Link
Ramachandran S, Lu H, Prabhu U, Ruoho AE. (2007) Purification and characterization of the guinea pig sigma-1 receptor functionally expressed in Escherichia coli. Protein Expr Purif 51 (2), 283-92. PubMed ID:16962337 | PMC Link
Blommel PG, Becker KJ, Duvbjak P, Fox BG. (2007) Enhanced bacterial protein expression during auto-induction obtained by alteration of lac repressor dosage and medium composition. Biotechnol Prog 23 (3), 585-98. PubMed ID: 17506520 | Search SBKB Publications portal | PMC Link
Brian G. Fox (email@example.com)
Transmembrane Protein Center
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Last edited:Mon 10 Mar 2014 - 5 years, 1 month ago