Biomaterial Database

Purification and characterization of recombinant Streptomyces clavuligerus isopenicillin N synthase produced in Escherichia coli. 1996

M Durairaj, and S E Jensen

Department of Biological Sciences, University of Alberta, Edmonton, Canada.

Recombinant isopenicillin N synthase from Streptomyces clavuligerus was produced in the form of inactive inclusion bodies in Escherichia coli. These inclusion bodies were solubilized by treatment with 5 M urea under reducing conditions. Optimization of refolding conditions to recover active isopenicillin N synthase indicated that a dialysis procedure carried out at a protein concentration of about 1.0 mg ml(-1) gave maximal recovery of active isopenicillin N synthase. Solubilized isopenicillin N synthase of more than 95% purity was obtained by passing this material through a DEAE-Trisacryl ion exchange column. Expression studies conducted at different temperatures indicated that isopenicillin N synthase was produced predominantly in a soluble, active form when expression was conducted at 20 degrees C, and accounted for about 20% of the total soluble protein. This high-level production facilitated the purification of soluble isopenicillin N synthase to near homogeneity in four steps. Characterization of the purified soluble and solubilized isopenicillin N synthase revealed that they are very similar.

UI	MeSH Term	Description	Entries
D007535	Isomerases	A class of enzymes that catalyze geometric or structural changes within a molecule to form a single product. The reactions do not involve a net change in the concentrations of compounds other than the substrate and the product.(from Dorland, 28th ed) EC 5.	Isomerase
D011994	Recombinant Proteins	Proteins prepared by recombinant DNA technology.	Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D002479	Inclusion Bodies	A generic term for any circumscribed mass of foreign (e.g., lead or viruses) or metabolically inactive materials (e.g., ceroid or MALLORY BODIES), within the cytoplasm or nucleus of a cell. Inclusion bodies are in cells infected with certain filtrable viruses, observed especially in nerve, epithelial, or endothelial cells. (Stedman, 25th ed)	Cellular Inclusions,Cytoplasmic Inclusions,Bodies, Inclusion,Body, Inclusion,Cellular Inclusion,Cytoplasmic Inclusion,Inclusion Body,Inclusion, Cellular,Inclusion, Cytoplasmic,Inclusions, Cellular,Inclusions, Cytoplasmic
D004926	Escherichia coli	A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.	Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D013302	Streptomyces	A genus of bacteria that form a nonfragmented aerial mycelium. Many species have been identified with some being pathogenic. This genus is responsible for producing a majority of the ANTI-BACTERIAL AGENTS of practical value.
D013696	Temperature	The property of objects that determines the direction of heat flow when they are placed in direct thermal contact. The temperature is the energy of microscopic motions (vibrational and translational) of the particles of atoms.	Temperatures
D046915	Penicillin-Binding Proteins	Bacterial proteins that share the property of binding irreversibly to PENICILLINS and other ANTIBACTERIAL AGENTS derived from LACTAMS. The penicillin-binding proteins are primarily enzymes involved in CELL WALL biosynthesis including MURAMOYLPENTAPEPTIDE CARBOXYPEPTIDASE; PEPTIDE SYNTHASES; TRANSPEPTIDASES; and HEXOSYLTRANSFERASES.	Penicillin-Binding Protein,Penicillin-Binding Protein 1,Penicillin-Binding Protein 1A,Penicillin-Binding Protein 1b,Penicillin-Binding Protein 2,Penicillin-Binding Protein 2a,Penicillin-Binding Protein 2b,Penicillin-Binding Protein 3,Penicillin-Binding Protein 4,Penicillin-Binding Protein 5,Penicillin-Binding Protein 6,Penicillin-Binding Protein 7,Penicillin-Binding Protein-2a,Peptidoglycan Synthetase,Penicillin Binding Protein,Penicillin Binding Protein 1,Penicillin Binding Protein 1A,Penicillin Binding Protein 1b,Penicillin Binding Protein 2,Penicillin Binding Protein 2a,Penicillin Binding Protein 2b,Penicillin Binding Protein 3,Penicillin Binding Protein 4,Penicillin Binding Protein 5,Penicillin Binding Protein 6,Penicillin Binding Protein 7,Penicillin Binding Proteins,Protein 1A, Penicillin-Binding,Protein 1b, Penicillin-Binding,Proteins, Penicillin-Binding,Synthetase, Peptidoglycan
D019751	Intramolecular Transferases	Enzymes of the isomerase class that catalyze the transfer of acyl-, phospho-, amino- or other groups from one position within a molecule to another. EC 5.4.	Mutases,Transferases, Intramolecular