Growth hormone from striped catfish (Pangasianodon hypophthalmus): genomic organization, recombinant expression and biological activity.

Growth hormone is an essential polypeptide required for normal growth and development of vertebrates. In this report, striped catfish (Pangasianodon hypophthalmus) growth hormone gene and cDNA were isolated by reverse transcriptase-polymerase chain reaction. The striped catfish growth hormone (scGH) encoding gene contains 5 exons and 4 introns. The cDNA sequence of the scGH gene contains a 603bp open reading frame and encodes for a 200-aa protein consisting of a putative 22-aa signal peptide and the mature 178-aa protein. The recombinant histidine-tagged scGH protein which expressed in Escherichia coli as inclusion bodies was unfolded, refolded and purified to near-homogeneity by Ni(2+)-NTA chromatography. Analysis of the secondary structure content by CD spectroscopy showed that the α-helical content of the refolded scGH is 55%. Elucidation of the folding pathway of scGH by fluorescence spectroscopy showed that denaturation transition of scGH is coincident and cooperative, consistent with the two-state denaturation mechanism. The purified scGH was biologically active and exhibited growth-promoting activity in striped catfish, but not tilapia.

A sequence-specific nicking endonuclease from streptomyces: purification, physical and catalytic properties.

A sequence-specific nicking endonuclease from Streptomyces designated as DC13 was purified to near homogeneity. Starting with 30 grams of wet cells, the enzyme was purified by ammonium sulfate fractionation, DEAE cellulose, and phenyl-Sepharose chromatography. The purified protein had a specific activity 1000 units/mg and migrated on SDS-PAGE gel with an estimated molecular weight of 71 kDa. Determination of subunit composition by gel filtration chromatography indicated that the native enzyme is a monomer. When incubated with different DNA substrates including pBluescript II KS, pUC118, pET-15b, and pET-26b, the enzyme converted these supercoiled plasmids to a mixture of open circular and linear DNA products, with the open circular DNA as the major cleavage product. Analysis of the kinetic of DNA cleavage showed that the enzyme appeared to cleave super-coiled plasmid in two distinct steps: a rapid cleavage of super-coiled plasmid to an open circular DNA followed a much slower step to linear DNA. The DNA cleavage reaction of the enzyme required Mg(2+) as a cofactor. Based on the monomeric nature of the enzyme, the kinetics of DNA cleavage exhibited by the enzyme, and cofactor requirement, it is suggested here that the purified enzyme is a sequence-specific nicking endonuclease that is similar to type IIS restriction endonuclease.

Adenosine deaminase from Streptomyces coelicolor: recombinant expression, purification and characterization.

The sequencing of the genome of Streptomyces coelicolor A3(2) identified seven putative adenine/adenosine deaminases and adenosine deaminase-like proteins, none of which have been biochemically characterized. This report describes recombinant expression, purification and characterization of SCO4901 which had been annotated in data bases as a putative adenosine deaminase. The purified putative adenosine deaminase gives a subunit Mr=48,400 on denaturing gel electrophoresis and an oligomer molecular weight of approximately 182,000 by comparative gel filtration. These values are consistent with the active enzyme being composed of four subunits with identical molecular weights. The turnover rate of adenosine is 11.5 s⁻¹ at 30 °C. Since adenine is deaminated ∼10³ slower by the enzyme when compared to that of adenosine, these data strongly show that the purified enzyme is an adenosine deaminase (ADA) and not an adenine deaminase (ADE). Other adenine nucleosides/nucleotides, including 9-ß-D-arabinofuranosyl-adenine (ara-A), 5'-AMP, 5'-ADP and 5'-ATP, are not substrates for the enzyme. Coformycin and 2'-deoxycoformycin are potent competitive inhibitors of the enzyme with inhibition constants of 0.25 and 3.4 nM, respectively. Amino acid sequence alignment of ScADA with ADAs from other organisms reveals that eight of the nine highly conserved catalytic site residues in other ADAs are also conserved in ScADA. The only non-conserved residue is Asn317, which replaces Asp296 in the murine enzyme. Based on these data, it is suggested here that ADA and ADE proteins are divergently related enzymes that have evolved from a common α/ß barrel scaffold to catalyze the deamination of different substrates, using a similar catalytic mechanism.