A second generation snp-derived Escherichia coli-Streptomyces shuttle expression vector that is generally transferable by conjugation

An Escherichia coli-Streptomyces shuttle vector (pJN100) was constructed, by inserting an origin of transfer (oriT), derived from the E. coli broad host range plasmid RK2, into pANT1202, a high-copy-number vector for gene expression in Streptomyces. The resulting conjugably transferable vector contains the pANT1202-derived SnpR (LysR-like protein) activated snpA promoter that drives strong heterologous expression of proteins. We initially demonstrated that plasmid pJN100 was transferred with high frequency (10(-5-7) exconjugants per recipient) into several Streptomyces strains that were refractory to transformation by other means. Plasmid pJN100 was also shown to be stable in E. coli and Streptomyces. We confirmed functional protein expression by using a pJN100 derivative to complement a mutant of Streptomyces griseus with a disrupted chromosomal copy of the gene nonM, a gene encoding an essential reductase in the nonactin biosynthesis gene cluster. High levels of protein expression were confirmed using Western blotting to assess the production of the serine esterase NonR, an enzyme responsible for nonactin resistance in the nonactin producer S. griseus.     

Studies on groundnut proteins. V. Physico-chemical properties of arachin prepared by different methods.

ORD,¹ CD, and fluorescence spectra of arachin prepared by Tombs' (Biochem. J., 96, 119; 1965), Dawson's (Anal. Biochem., 41, 305; 1971) or Shetty and Rao's (Anal. Biochem., 62, 108; 1974) procedure were measured; the effect of denaturants such as SDS, GuHCl, and acid was also determined. ORD and CD spectra showed differences, whereas fluorescence spectra did not show any difference. The effect of the denaturants was the same on the three arachins. At low concentrations of GuHCl (<2 m), the denaturant was bound by the protein molecule without causing any conformational change. The binding affinity varied among the arachins.     

Studies on the Hemolytic Properties of Protamine

The basic protein protamine causes a rapid hemolysis when incubated with the red blood cells of many mammalian species. The age of the cells does not affect the process. Neutralization of the active side groups of the protamine molecule with formalinization demonstrates that a specific degree of charge is necessary for hemolysis, as more than 30 per cent of the guanidine groups must remain unreacted to maintain activity. Unlike the hemolysis induced by the synthetic polypeptides polylysine and polyhomoarginine, protamine hemolysis is temperature-dependent. Whole lipoprotein material derived from red blood cell membranes inhibits protamine hemolysis to a greater extent than do the membranes themselves, serum, serum protein fractions, or cholesterol. The phosphatide and protein moieties derived from the membranes are quite avid in inhibiting protamine hemolysis. A probable explanation is that intracellular aggregation of these structural elements may cause changes in electrostatic charge and surface tension which result in increased permeability. The hemolytic and antitumor cell properties of protamine could not be segregated from its animal toxicity. Despite formalinization to a degree which eliminated the former, the compound remained quite toxic to mice and rabbits.     

Studies of mammalian glucoside conjugation

The mammalian glucoside-conjugation pathway was studied by using p-nitrophenol as the model substrate and mouse liver microsomal preparations as the source of enzyme. The microsomal preparations supplemented with UDP-glucose glucosylated p-nitrophenol; p-nitrophenyl glucoside was identified by chromatography in six solvent systems. The unsolubilized glucosyltransferase of fresh microsomal preparations did not follow the usual Michaelis-Menten kinetics and was easily inhibited by many steroids. All the steroids tested inhibited glucosylation of p-nitrophenol to a greater degree than glucuronidation of p-nitrophenol when assayed in the same microsomal preparations. The steroids inhibited glucosylation with the following decreasing effectiveness: pregnan-3alpha-ol-20beta-one (3alpha-hydroxypregnan-20-beta-one)>oestradiol-17beta 3-methyl ether>oestradiol-17beta>oestriol>pregnane-3alpha,20beta-diol>oestrone. Pregnan-3alpha-ol-20beta-one, pregnane-3alpha,20beta-diol and oestrone had negligible effect on glucuronidation.