RAMA stain: a fast, sensitive and less protein-modifying CBB R250 stain.

SDS-PAGE and CBB staining are two of the most popular methods used for protein analysis. Although many reports that describe such staining methods have been published, these conventional protocols require several hours or days for staining and de-staining. In this study we describe a recently developed, fast and sensitive CBB staining method that utilizes the staining solution of RAMA that consists of the low-cost reagents: CBB R250, acetic acid, methanol and ammonium sulfate, and the destaining solution of water. Our method dose dependently detects 12 nanograms protein within 60 min and with a wide protein spectrum. Although the features of the dose-dependent relationship depend upon protein amounts and protein types, for most of the protein samples tested, a linear relationship was observed in the region from 12 to 330 ng. Moreover, through further washing, the detection sensitivity of protein is enhanced and reaches a maximum at 1.4 ng and then gradually decreases in the de-staining process. It has been shown recently through MS analyses that the sensitive colloidal CBB staining methods frequently result in artifactual methylations due to the strong acid and long contact during staining and the destaining processes. Such artifacts were reported to be reduced by the replacement of strong inorganic acid with acetic acid and because RAMA utilizes acetic acid and is in contact with the proteins for a short time during staining and de-staining, it is expected that in vitro artifacts will be reduced. Finally, MS analyses of RAMA-stained protein bands were revealed not to have been methylated.

[Production of recombinant human CRP and its application on clinical testing].

Recently, changes in the serum CRP level 1/10 the concentration range ordinarily used as a marker of acute inflammation has received attention in relation to cardiovascular injury at the AACC/CDC joint forum at Atlanta held on March 13, 2001. We have succeeded in the development of recombinant human CRP(rCRP) by inserting the cloned CRP gene into expression vector pTRP, followed by transformation of E. coli. Genes encoding the signal peptide of E. coli alkaline phosphatase and kil gene were additionally inserted, so that rCRP can be secreted into the culture supernatant. Five grams of rCRP was purified from 180 L culture supernatant by affinity chromatography. The purified rCRP was indistinguishable from native rCRP with respect to Ca(2+)-dependent binding activity to phosphorylcholine, electrophoretic behavior in the presence or absence of SDS, N-terminal amino acid, and immunochemical properties. rCRP was found to have a potential as a reference material and/or calibrator for hsCRP assay.

Secretory production of recombinant human C-reactive protein in Escherichia coli, capable of binding with phosphorylcholine, and its characterization.

Recombinant human CRP (rhCRP) was secreted into culture supernatant of Escherichia coli by co-expressing kil gene that has a function to secrete colicin E1 outside the cell. Highly purified 5 g rhCRP was produced from 180 L culture supernatant by affinity chromatography. The purified rhCRP was indistinguishable from the native one with respect to Ca(2+)-dependent binding ability to phosphorylcholine, electrophoretic behavior, N-terminal amino acid analysis, and immunochemical properties. The molecular weight of rhCRP monomer was determined to be 23059.7 Da by TOF/MS analysis. These results indicate that rhCRP has the same protein structure as native one and that rhCRP has the potential as a reference material and/or calibrator of high-sensitivity CRP assay to predict the risk of cardiovascular disease.

Characterization and molecular cloning of a novel enzyme, inorganic polyphosphate/ATP-glucomannokinase, of Arthrobacter sp. strain KM.

A bacterium exhibiting activities of several inorganic polyphosphate [poly(P)]- and ATP-dependent kinases, including glucokinase, NAD kinase, mannokinase, and fructokinase, was isolated, determined to belong to the genus Arthrobacter, and designated Arthrobacter sp. strain KM. Among the kinases, a novel enzyme responsible for the poly(P)- and ATP-dependent mannokinase activities was purified 2,200-fold to homogeneity from a cell extract of the bacterium. The purified enzyme was a monomer with a molecular mass of 30 kDa. This enzyme phosphorylated glucose and mannose with a high affinity for glucose, utilizing poly(P) as well as ATP, and was designated poly(P)/ATP-glucomannokinase. The K(m) values of the enzyme for glucose, mannose, ATP, and hexametaphosphate were determined to be 0.50, 15, 0.20, and 0.02 mM, respectively. The catalytic sites for poly(P)-dependent phosphorylation and ATP-dependent phosphorylation of the enzyme were found to be shared, and the poly(P)-utilizing mechanism of the enzyme was shown to be nonprocessive. The gene encoding the poly(P)/ATP-glucomannokinase was cloned from Arthrobacter sp. strain KM, and its nucleotide sequence was determined. This gene contained an open reading frame consisting of 804 bp coding for a putative polypeptide with a calculated molecular mass of 29,480 Da. The deduced amino acid sequence of the polypeptide exhibited homology to the amino acid sequences of the poly(P)/ATP-glucokinase of Mycobacterium tuberculosis H37Rv (level of homology, 45%), ATP-dependent glucokinases of Corynebacterium glutamicum (45%), Renibacterium salmoninarum (45%), and Bacillus subtilis (35%), and proteins of bacteria belonging to the order Actinomyces whose functions are not known. Alignment of these homologous proteins revealed seven conserved regions. The mannose and poly(P) binding sites of poly(P)/ATP-glucomannokinase are discussed.