Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.6 (thromboplastin)
 13,278 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Using the maltose-binding protein (MBP) fusion vector pMAL-c1 from C. V. Maina et al. (1988, Gene 74, 365-373), we have constructed expression vectors which contain a sequence encoding six consecutive histidine residues (His6-tag) at the 3' end of the MBP-encoding malE gene which is followed by either a thrombin-binding site (LVPRGS) or a factor Xa-binding site (IEGR). The benefits of this approach include; (a) high expression levels of soluble MBP fusion proteins (exceeding 2% of the total cellular protein), (b) high-quality purification of proteins under various conditions (high salt, low salt, denaturing, nondenaturing, etc.), and (c) two alternative protease cleavage sites to test for the most efficient cleavage of each fusion protein. We also constructed these MBP-His 6-tag expression vectors with alternative selection markers (Ampr, Kanr) and alternative promoters (tac, T7). Using these constructs, we expressed and purified several proteins of which we present two, penicillin-binding protein PBP2a and UDP-N-acetylmuramate:L-alanine ligase (MurC), and compare their expression level and purity with other expression systems. We also discuss the use of minimal media with supplements versus rich media and cell growth strategies to optimize the protein yield in general and for isotope labeling.
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PMID:High-level expression of soluble protein in Escherichia coli using a His6-tag and maltose-binding-protein double-affinity fusion system. 926 77