Improved Purification Protocol Research Articles

Improved purification protocol of a Fusarium solani pisi recombinant cutinase by phase partitioning in aqueous two-phase systems of polyethylene glycol and phosphate

Recombinant Fusarium solani pisi cutinase is generally purified by a multistep process that includes (a) osmotic shock, (b) acid precipitation, and chromatography. Steps a and b provide a combined yield of only 77% with a purification factor of 4.1. We report a single-step process using an aqueous two-phase partitioning system of 25% (w/w) polyethylene glycol 1000 and 10% (w/w) sodium phosphate, pH 4.8, to obtain 91% yield with a purification factor of 4.1. This protocol is completed within 20 min, is a cheap alternative to steps a and b, and is easy to scale up.

The primary structure of the glutamic acid-specific protease of Streptomyces griseus

The amino acid sequence and part of the DNA sequence of a glutamic acid-specific serine protease from Streptomyces griseus is reported. This protease is shown to be homologous with other serine proteases. An improved purification protocol for this enzyme is described.

Purification of flavanone 3β-hydroxylase from Petunia hybrida: Antibody preparation and characterization of a chemogenetically defined mutant

Flavanone 3β-hydroxylase from Petunia hybrida has been purified to apparent homogeneity utilizing an improved purification protocol including chromatography of the partially purified enzyme on hydroxyapatite, chromatofocusing, and hydrophobic interaction chromatography on phenyl-Superose. The specificity of mouse and rabbit polyclonal antisera directed to flavanone 3β-hydroxylase was demonstrated by Western blotting and immunotitration with crude extracts from wild-type flowers of P. hybrida and with the purified enzyme. Cross-reactivity was observed with flavanone 3β-hydroxylase from extracts of illuminated parsley cells. A Petunia mutant with white flowers, previously shown to lack 3β-hydroxylase activity and to accumulate flavanone glycosides, showed complete absence of the enzyme protein.

Substrate specificity and variables affecting efficiency of mammalian flavin adenine dinucleotide synthetase

Substrate specificity and product inhibition have been evaluated by using purified rat liver FAD synthetase (ATP:FMN adenylyltransferase, EC 2.7.7.2), obtained by an improved purification protocol with optimized flavin affinity chromatography. FMN analogues studied fall into three general classifications: those with substitution on the pyrimidinoid ring and nitrogen replacement, those with substitution on the benzenoid ring, and those with N(10) side chain modifications. Substitutions on the pyrimidinoid ring and replacement of nitrogens have the greatest influence on binding to enzyme and FAD formation. When the hydrogen-bonding capacity of the NH group at position 3 is blocked or removed by substitution, such FMN analogues do not act as substrates or inhibitors of the enzyme. Substitutions on the benzenoid ring by small groups seem to be tolerated, while larger groups inhibit binding. Length of the N(10) side chain is optimal with five carbons and has greatest affinity for the natural ribityl side chain. Affinity matrices show similar binding characteristics in that the N(3)-(carboxymethyl)riboflavin-agarose does not bind enzyme, while agaroses linked to the flavin N(10) side chain provide varying degrees of purification. The C = O group at position 2, the NH group at position 3, and a five-carbon side chain at the N(10) position seem to be most crucial for flavin substrate binding to enzyme. Nucleoside triphosphates other than ATP do not act as substrates or inhibitors when sufficient Mg2+ is present. Products of the reaction, FAD and PPi, act as inhibitors against both ATP and FMN.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the human plasma kallikrein-kinin system: α-kallikrein does not directly activate blood neutrophils

α-Kallikrein was prepared using an improved purification protocol (Burger, D., Schleuning, W.-D. and Schapira, M. (1986) J. Biol. Chem. 261, 324–327) and was employed to reevaluate our previous observations indicating that kallikrein activates blood neutrophils by a mechanism requiring an uncleaved M r 52,000 NH 2- terminal heavy chain. Cellular activation was evaluated by measuring neutrophil aggregation, release of both vitamin B 12 binding capacity and myeloperoxidase, and generation of superoxide anion. Whereas all these indicators were evoked by exposing neutrophils to f-Met-Leu-Phe, phorbol myristate acetate, zymosan activated serum, or the calcium ionophore A 23187, we show here that neutrophils incubated with α-kallikrein remained unactivated. Moreover, we demonstrate that this lack of activation is accompanied by an inability of neutrophils to specifically bind α-kallikrein.

Studies on the interaction of firefly luciferase with triazine dyes

The interaction of firefly luciferase with several triazine dyes has been investigated. Of those tested, the anthraquinone dye, Procion blue MX-R, displayed relatively high affinity [dissociation constant ( K D)  5μ M] and irreversibly inactivated the enzyme in a time dependent fashion. The substrates, MgATP (ATP is adenosine triphosphate) and luciferin, protected the enzyme against inactivation with Procion blue MX-R and following quantitative inactivation, incorporation of 1 mol dye per mol enzyme [molecular weight ( M r)  50 000] was observed. It is suggested that Procion blue MX-R binding is active site-directed and that the dye binding traverses both MgATP and luciferin binding sites. These data are exploited to evolve an improved purification protocol for firefly luciferase.

Improved Purification Protocol for Oxalate Oxidase from Barley Roots

Abstract A purification scheme is described for oxalate oxidase from barley roots. This procedure involves gel filtration on Bio-Gel A 0.5 m and affinity chromatography using oxalate, immobilized on Affi-Gel 10, as a ligand. The purified enzyme was homogeneous on SDS-gel electrophoresis, thin-layer electrophoresis and high-performance liquid chromatography (HPLC). The molecular weight of the enzyme was 150.000 by SDS-gel electrophoresis and the specific activity was 1.9 U/mg protein.

An improved purification protocol for ribulose 1,5-bisphosphate carboxylase from Chromatium vinosum

An improved purification protocol for ribulose 1,5-bisphosphate carboxylase from Chromatium vinosum