Use of a coupled transcriptional system for consistent overexpression and purification of UDG-Ugi complex and Ugi from Escherichia coli

We have designed a novel coupled transcriptional construct wherein Escherichia coil uracil DNA glycosylase (UDC:) and Bacillus subtilis phage PBS-2 encoded uracil DNA glycosylase inhibitor protein (Ugi) genes were cloned in tandem, downstream of an inducible promoter (P-trc). Use of this bicistronic operon has allowed purification of large amounts of UDG-Ugi complex formed in vivo. The system has also been exploited for purification of large amounts of Ugi. While establishing the expression system, one of the constructs showed detectable suppression of UAG termination codon and resulted in accumulation of a minor population of a putative readthrough polypeptide cor responding to UDG. We discuss the likely occurrence of such a phenomenon in overproduction of other recombinant proteins. Finally, the usefulness of the operon construct in convenient mutational analysis to study the mechanism of UDG-Ugi interaction is also discussed.

The homologous recombination system of phage lambda. Pairing activities of beta protein

The red genes of phage lambda specify two proteins, exonuclease and beta protein, which are essential for its general genetic recombination in recA- cells. These proteins seem to occur in vivo as an equimolar complex. In addition, beta protein forms a complex with another polypeptide, probably of phage origin, of Mr 70,000. The 70-kDa protein appears to be neither a precursor nor an aggregated form of either exonuclease or beta protein, since antibodies directed against the latter two proteins failed to react with 70-kDa protein on Ouchterlony double diffusion analysis. beta protein promotes Mg2+-dependent renaturation of complementary strands (Kmiec, E., and Holloman, W. K. (1981) J. Biol. Chem. 256, 12636-12639). To look for other pairing activities of beta protein, we developed methods of purification to free it of associated exonuclease. Exonuclease-free beta protein appeared unable to cause the pairing of a single strand with duplex DNA; however, like Escherichia coli single strand binding protein (SSB), beta protein stimulated formation of joint molecules by recA protein from linear duplex DNA and homologous circular single strands. Like recA protein, but unlike SSB, beta protein promoted the joining of the complementary single-stranded ends of phage lambda DNA. beta protein specifically protected single-stranded DNA from digestion by pancreatic DNase. The half-time for renaturation catalyzed by beta protein was independent of DNA concentration, unlike renaturation promoted by SSB and spontaneous renaturation, which are second order reactions. Thus, beta protein resembles recA protein in its ability to bring single-stranded DNA molecules together and resembles SSB in its ability to reduce secondary structure in single-stranded DNA.

Expression, Purification and Characterization of Peanut (Arachis hypogaea) Agglutinin (PNA) from Baculovirus Infected Insect Cells

Peanut (Arachis hypogaea) seed lectin, PNA is widely used to identify tumor specific antigen (T-antigen), Gal beta 1-3GalNAc on the eukaryotic cell surface. The functional amino acid coding region of a cDNA clone, pBSH-PN was PCR amplified and cloned downstream of the polyhedrin promoter in the Autographa californica nucleopolyhedrovirus (AcNPV) based transfer vector pVL1393. Co-transfection of Spodoptera frugiperda cells (Sf9) with the transfer vector, pAcPNA and AcRP6 (a recombinant AcNPV having B-gal downstream of the polyhedrin promoter) DNAs produced a recombinant virus, AcPNA which expresses PNA. Infection of suspension culture of Sf9 cells with plaque purified AcPNA produced as much as 9.8 mg PNA per liter (2.0 x 10(6) cells/ml) of serum-free medium. Intracellularly expressed protein (re-PNA) was purified to apparent homogeneity by affinity chromatography using ECD-Sepharose. Polyclonal antibodies against natural PNA (n-PNA) crossreacted with re-PNA. The subunit molecular weight (30 kDa), hemagglutination activity, and carbohydrate specificity of re-PNA were found to be identical to that of n-PNA, thus confirming the abundant production of a functionally active protein in the baculovirus expression system.

Purification and functional characterization of type II DNA topoisomerase from rat testis and comparison with topoisomerase II from liver

A number of studies in yeast have shown that DNA topoisomerase TI is essential for chromosome condensation and disjunction during mitosis at the metaphase/anaphase transition and meiosis I. Accordingly, kinetic and mechanistic studies have implied a role for topoisomerase rr in chromosome disjunction. As a step toward understanding the nature and role of topoisomerase II in a mammalian germline in vivo, we have purified topoisomerase II from rat testis to homogeneity and ascertained several of its catalytic activities in conjunction with that of the purified enzyme from liver. The purified enzymes appeared to be monomers under denaturing conditions; however, they differed in their relative molecular mass. Topoisomerase II from testis and liver have apparent molecular masses of 150 +/- 10 kDa and 160 +/- 10 kDa, respectively. The native molecular mass of testis topoisomerase II as assayed by immunoblot analysis of cell-foe extracts, prepared in the presence of SDS and a number of protease inhibitors, corroborated with the size of the purified enzyme. Both enzymes are able to promote decatenation and relax supercoiled DNA substrates in an ATP and Mg2+-dependent manner. However, quantitative comparison of catalytic properties of topoisomerase II from testis with that of the enzyme from liver displayed significant differences in their efficiencies. Optimal pH values for testis enzyme are 6.5 to 8.5 while they are 6 to 7.5 for the liver enzyme. Intriguingly, the relaxation activity of liver topoisomerase II was inhibited by potassium glutamate at 1 M, whereas testis enzyme required about half its concentration. These findings argue that topoisomerase II from rat testis is structurally distinct from that of its somatic form and the functional differences between the two enzymes parallels with the physiological environment that is unique to these two tissues.

Purification, properties and synthesis of delta-aminolaevulinate dehydratase from Neurospora crassa.

Delta-aminolaevulinate dehydratase, the second and rate-limiting enzyme of the haem-biosynthetic pathway, was purified 300-fold from induced cultures of Neurospora crassa. The native enzyme has a mol.wt. of about 350000, whereas the salt-treated enzyme after incubation at 37 degrees C for 10 min has a mol.wt. of about 232000. The mol.wt. of the subunit is about 38000. Antibodies to the purified enzyme were raised in rabbits. By using radiolabelling and immunoprecipitation techniques it was shown that addition of iron and laevulinate to iron-deficient cultures brings about a significant increase in the synthesis of the enzyme, and protoporphyrin, the penultimate end product of the pathway, represses enzyme synthesis.

Role of Protozoa in the Aerobic Purification of Sewage

DURING recent years, there has been increasing amount of evidence regarding the importance of protozoa in the aerobic purification of sewage1–4 and, more recently, some quantitative observations on protozoa in determining the condition of sludge and quality of effluent have been recorded5–10. The evidence so far obtained has, however, been only indirect, chiefly owing to the difficulty in separating the protozoa from the associated bacteria. This has now been achieved and the object of this note is to show that the isolated protozoa can bring about practically all the changes associated with the purification. The part played by the bacteria is almost negligible.

Enzymic Conversion of Agmatine to Putrescine inL athyrms satiuus seedlings - Purification and properties of a multifunctional enzyme (putrescine synthase)

The participation of a multifunctional enzy(am sein - gle polypeptide with multiple catalytic activities (14)) has been demonstrated in the conversion of agmatine to putrescine in Lathyrus sativus seedlings. This enzyme (putrescine synthase) with inherent activities of agmatine iminohydrolase, putrescine transcarbamylase, ornithine transcarbamylase, and carbamate has been purified to homogeneity anhda s M, = 55,000.

Purification of a bacterial organophosphate-hydrolysing phosphatase by cibacron 3GA-Sepharose affinity chromatography

A phosphatase catalysing the hydrolysis of organophosphorus pesticides was purified to homogeneity using Cibacron 3GA-Sepharose CL 6B affinity chromatography. The enzyme which is localized in the periplasm of the bacterium Image NC5 was extracted by treating with 0.2M MgCl2, pH 8.4. The enzyme was adsorbed to the Cibacron-Sepharose at pH 7.0 and eluted with Tris-HCl buffer at pH 8.0, with 47 per cent recovery. The enzyme thus obtained was electrophoretically homogeneous. This simple affinity purification procedure enhances the potential for its use in large scale detoxification systems.

Purification and properties of synephrinase from Arthrobacter synephrinum

Synephrinase, an enzyme catalyzing the conversion of (−)-synephrine into p-hydroxyphenylacetaldehyde and methylamine, was purified to apparent homogeneity from the cell-free extracts of Arthrobacter synephrinum grown on (±)-synephrine as the sole source of carbon and nitrogen. A 40-fold purification was sufficient to produce synephrinase that is apparently homogeneous as judged by native polyacrylamide gel electrophoresis and has a specific activity of 1.8 μmol product formed /min/mg protein. Thus, the enzyme is a relatively abundant enzyme, perhaps comprising as much as 2.5% of the total protein. The enzyme essentially required a sulfhydryl compound for its activity. Metal ions like Mg2+, Ca2+, and Mn2+ stimulated the enzyme activity. Metal chelating agents, thiol reagents, denaturing agents, and metal ions like Zn2+, Hg2+, Ag1+, and Cu2+ inhibited synephrinase activity. Apart from (−)-synephrine, the enzyme acted upon (±)-octopamine and β-methoxysynephrine. Molecular oxygen was not utilized during the course of the reaction. The molecular mass of the enzyme as determined by Sephadex G-200 chromatography, was around 156,000. The enzyme was made up of four identical subunits with a molecular mass of 42,000.

Partial purification and properties of a transamidinase from Lathyrus sativus seedlings. Involvement in homoarginine metabolism and amine interconversions

A transamidinase was purified 463-fold from Lathyrus sativus seedlings by affinity chromatography on homoarginine--Sepharose. The enzyme exhibited a wide substrate specificity, and catalysed the reversible transfer of the amidino groups from donors such as arginine, homoarginine and canavanine to acceptors such as lysine, putrescine, agmatine, cadaverine and hydroxylamine. The enzyme could not be detected in the seeds, and attained the highest specific activity in the embryo axis on day 10 after seed germination. Its thiol nature was established by strong inhibition by several thiol blockers and thiol compounds in the presence of ferricyanide. In the absence of an exogenous acceptor, it exhibited weak hydrolytic activity towards arginine. It had apparent mol.wt. 210000, and exhibited Michaelis--Menten kinetics with Km 3.0 mM for arginine. Ornithine competitively inhibited the enzyme, with Ki 1.0 mM in the arginine--hydroxylamine amidino-transfer reaction. Conversion experiments with labelled compounds suggest that the enzyme is involved in homoarginine catabolism during the development of plant embryo to give rise to important amino acids and amine metabolites. Presumptive evidence is also provided for its involvement in the biosynthesis of the guanidino amino acid during seed development. The natural occurrence of arcain in L. sativus and mediation of its synthesis in vitro from agmatine by the transamidinase are demonstrated.

Enzyme-catalysed non-oxidative decarboxylation of aromatic acids: I.Purification and spectroscopic properties of 2,3 dihydroxybenzoic acid decarboxylase from Image Image

In order to understand the molecular mechanism of non-oxidative decarboxylation of aromatic acids observed in microbial systems, 2,3 dihydroxybenzoic acid (DHBA) decarboxylase from Image Image was purified to homogeneity by affinity chromatography. The enzyme (Mr 120 kDa) had four identical subunits (28 kDa each) and was specific for DHBA. It had a pH optimum of 5.2 and Km was 0.34mM. The decarboxylation did not require any cofactors, nor did the enzyme had any pyruvoyl group at the active site. The carboxyl group and hydroxyl group in the Image -position were required for activity. The preliminary spectroscopic properties of the enzyme are also reported.

Studies on the purification of banana polyphenoloxidase

Studies on the extractability of polyphenoloxidase (PPO) from the pulp of five banana cultivars revealed a varietal difference in the nature of binding of the PPO in the cell, with the enzyme being entirely in the soluble fraction in one and partly associated with the cell wall in others, necessitating use of a detergent to release it from the latter. Partial purification by acetone precipitation and chromatography using a DEAE-cellulose column yielded two major fractions DE-I and DE-II with purifications of 4- and 16·3-fold and activity recoveries of 38·2 and 43·3% respectively. Further gel filtration of the two fractions on a Sephadex G-100 column improved the purifications to 44- and 50-fold respectively with full activity recovery. Polyacrylamide gel electrophoretic studies showed the two fractions to be composed of isoenzymes differing in pattern. The purified enzyme showed maximum absorption at 275 nm.

Purification and Characterization of a New Indole Oxygenase from the Leaves of Tecoma stans L.

A new indole oxygenase from the leaves of Tecoma stans was isolated and purified to homogenity. The purified enzyme system catalyzes the conversion of indole to anthranilic acid. It is optimally active at pH 5.2 and 30°C. Two moles of oxygen are consumed and one mole of anthranilic acid is formed for every mole of indole oxidized. Dialysis resulted in complete loss of the activity. The inactive enzyme could be reactivated by the addition of concentrated dialysate. The enzyme is not inhibited by copperspecific chelators, non-heme iron chelators or atebrin. It is not a cuproflavoprotein, unlike the other indole oxygenases and oxidases.

Purification of clofibrate-induced carnitine acetyltransferase from rat liver mitochondria

The enzyme carnitine acetyltransferase (acetyl-CoA:carnitine O-acetyltransferase, EC 2.3.1.7) has been purified to homogeneity from hepatic mitochondria of clofibrate-fed rats. It is a protein of molecular weight 56 000 composed of two non-identical subunits of molecular weight 34 000 and 25 000. The enzyme is inhibited by palmityl-CoA as well as acetyl carnitine. The inhibition by fatty acyl-CoA is competitive with respect to both the substrates, carnitine and acetyl-CoA. The inhibition by acetylcarnitine is reversed by carnitine but not by acetyl-CoA.