Biomedical subjects

M Durairaj

Publications and source records attributed to M Durairaj.

At least 19 recordsLinked to original sources

PubMed1996

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

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.

Escherichia coli↗

PubMed1996

Genetic and biochemical analysis of the cysteinyl residues of isopenicillin N synthase from Streptomyces clavuligerus.

Isopenicillin N synthase (IPNS) from Streptomyces clavuligerus catalyses the oxidative cyclization of the acyclic tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine into isopenicillin N. All four of the cysteine residues found in this enzyme were mutated individually into serine residues, either by the polymerase chain reaction or by single-strand site-directed mutagenesis. Functional analysis of these single mutants showed that the C104S mutant lost more than 96% of its activity, while the remaining C37S, C142S, and C251S mutants each lost 30-50% of their activity. Treatment with the thiol-group-specific reagent N-ethylmaleimide confirmed the importance of the cysteine 104 residue. Activity analysis of an IPNS triple mutant (C37S, C142S, and C251S), prepared by recombining fragments of the IPNS-encoding pcbC gene from each of the three single mutants, showed that it had lost more than 90% of its activity. Conformational analysis by circular dichroism spectroscopy indicated that the IPNS triple mutant was structurally different from the wild type, suggesting that the loss of activity may be due to conformational changes rather than active site modifications.

Base Sequence↗

PubMed1992

High-level expression of the Streptomyces clavuligerus isopenicillin N synthase gene in Escherichia coli.

The pcbC gene, which encodes isopenicillin N synthase (IPNS), was subcloned from Streptomyces clavuligerus into Escherichia coli by using the pT7 series of plasmid vectors. The polymerase chain reaction was used to introduce an NdeI site at the translation initiation codon of pcbC, allowing the gene to be inserted behind an E. coli type of ribosome binding site. This construction directed high-level expression of IPNS, but the IPNS was in an inactive form in inclusion bodies. Active IPNS was recovered by solubilizing and renaturing the protein.

Base Sequence↗

PubMed1983

Pulmonary hypertension in isolated secundum atrial septal defect: high frequency in young patients.

Out of 709 consecutive patients with isolated secundum atrial septal defect, the pulmonary artery systolic pressure was greater than 50 mm Hg in 118 patients (17%). Pulmonary hypertension was present in 13% of patients under 10 years and in 14% aged 11 to 20 years. The Eisenmenger reaction was present in 9% of the 709 patients. The frequency of the Eisenmenger reaction was high in young patients and was not significantly different in patients in the first and second decades as compared to older patients. None of our patients with pulmonary hypertension resided at high altitude. The high frequency of pulmonary hypertension in our young patients cannot be satisfactorily explained. Autopsy studies suggest that in some, pulmonary hypertension is due to the persistence of the fetal pulmonary vascular pattern.

Adolescent↗

PubMed1981