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Overproduction and one-step purification of Escherichia coli UDP-N-acetylglucosamine enolpyruvyl reductase.
Protein Expr Purif. 1995 Dec; 6(6):757-62.PE

Abstract

The Escherichia coli gene murB, encoding the enzyme uridine-5'-diphospho-N-acetyl-2-amino-2-deoxy-3-O-lactylglucosenicoti namide adenine dinucleotide phosphate oxidoreductase (EC 1.1.1.158) (EP-reductase), the second enzyme in the peptidoglycan biosynthetic pathway, has been amplified using PCR technology with the Kohara recombinant lambda phage E11C11 (534) as template. The synthetic gene was subcloned into the NdeI and BamHI restriction sites of the expression vector pT7-7, designed to utilize T7 RNA polymerase to direct transcription of the target gene, in a two-step procedure. The first step involved the directional insertion of the 590-bp NdeI to BamHI restriction fragment of murB into the pT7-7 vector to give the plasmid pT7-7-murB-590. The construction of the desired overproducing plasmid was completed by the bidirectional insertion of the 442-bp BamHI to BamHI restriction fragment of murB into a similarly restricted pT7-7-murB-590 plasmid followed by restriction digestion to select the properly oriented insert, pT7-7-murB. Overexpression of EP-reductase from the E. coli strain BL 21 (DE 3) containing the pT7-7-murB gene, after induction, allowed the production of 36 mg of target protein per 3 wet grams of E. coli cells. The EP-reductase was purified in a single step utilizing dye-ligand chromatography to yield 30 mg of pure protein. The availability of these levels of reductase will allow the mechanism of this pivotal enzyme to be thoroughly studied as a potential target for the design of a new generation of antibiotics. In addition, the EP-reductase generated in this study has been utilized as a coupling enzyme to assay the first enzyme in the peptidoglycan biosynthetic pathway, UDP-N-acetylglucosamine enolpyruvyl transferase, and these results are also presented.

Authors+Show Affiliations

Interdepartmental Program in Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor 48109-1065, USA.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

8746627

Citation

Tayeh, M A., et al. "Overproduction and One-step Purification of Escherichia Coli UDP-N-acetylglucosamine Enolpyruvyl Reductase." Protein Expression and Purification, vol. 6, no. 6, 1995, pp. 757-62.
Tayeh MA, Dotson GD, Clemens JC, et al. Overproduction and one-step purification of Escherichia coli UDP-N-acetylglucosamine enolpyruvyl reductase. Protein Expr Purif. 1995;6(6):757-62.
Tayeh, M. A., Dotson, G. D., Clemens, J. C., & Woodard, R. W. (1995). Overproduction and one-step purification of Escherichia coli UDP-N-acetylglucosamine enolpyruvyl reductase. Protein Expression and Purification, 6(6), 757-62.
Tayeh MA, et al. Overproduction and One-step Purification of Escherichia Coli UDP-N-acetylglucosamine Enolpyruvyl Reductase. Protein Expr Purif. 1995;6(6):757-62. PubMed PMID: 8746627.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Overproduction and one-step purification of Escherichia coli UDP-N-acetylglucosamine enolpyruvyl reductase. AU - Tayeh,M A, AU - Dotson,G D, AU - Clemens,J C, AU - Woodard,R W, PY - 1995/12/1/pubmed PY - 1995/12/1/medline PY - 1995/12/1/entrez SP - 757 EP - 62 JF - Protein expression and purification JO - Protein Expr Purif VL - 6 IS - 6 N2 - The Escherichia coli gene murB, encoding the enzyme uridine-5'-diphospho-N-acetyl-2-amino-2-deoxy-3-O-lactylglucosenicoti namide adenine dinucleotide phosphate oxidoreductase (EC 1.1.1.158) (EP-reductase), the second enzyme in the peptidoglycan biosynthetic pathway, has been amplified using PCR technology with the Kohara recombinant lambda phage E11C11 (534) as template. The synthetic gene was subcloned into the NdeI and BamHI restriction sites of the expression vector pT7-7, designed to utilize T7 RNA polymerase to direct transcription of the target gene, in a two-step procedure. The first step involved the directional insertion of the 590-bp NdeI to BamHI restriction fragment of murB into the pT7-7 vector to give the plasmid pT7-7-murB-590. The construction of the desired overproducing plasmid was completed by the bidirectional insertion of the 442-bp BamHI to BamHI restriction fragment of murB into a similarly restricted pT7-7-murB-590 plasmid followed by restriction digestion to select the properly oriented insert, pT7-7-murB. Overexpression of EP-reductase from the E. coli strain BL 21 (DE 3) containing the pT7-7-murB gene, after induction, allowed the production of 36 mg of target protein per 3 wet grams of E. coli cells. The EP-reductase was purified in a single step utilizing dye-ligand chromatography to yield 30 mg of pure protein. The availability of these levels of reductase will allow the mechanism of this pivotal enzyme to be thoroughly studied as a potential target for the design of a new generation of antibiotics. In addition, the EP-reductase generated in this study has been utilized as a coupling enzyme to assay the first enzyme in the peptidoglycan biosynthetic pathway, UDP-N-acetylglucosamine enolpyruvyl transferase, and these results are also presented. SN - 1046-5928 UR - https://www.unboundmedicine.com/medline/citation/8746627/Overproduction_and_one_step_purification_of_Escherichia_coli_UDP_N_acetylglucosamine_enolpyruvyl_reductase_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S1046-5928(85)70006-X DB - PRIME DP - Unbound Medicine ER -