Protein and Peptide Letters - Volume 12, Issue 7, 2005

Arginine is finding a wide range of applications in production of proteins. Arginine has been used for many years to assist protein refolding. This effect was ascribed to aggregation suppression by arginine of folding intermediates during protein refolding. Recently, we have observed that arginine facilitates elution of antibodies during Protein-A chromatography and solubilizes insoluble proteins from inclusion bodies, which Read More

Enzymatic peptide syntheses may be either thermodynamically- or kinetically-controlled. The former may be catalyzed by any proteases; the latter is limited to serine and cysteine proteases. This methodology is quite stereospecific and avoids side chain protection but is suffering of some drawbacks. Thus, only two industrial processes are by now developed: the production of aspartame and the conversion of porcine into h Read More

We have developed a simple method for preparing a tagged protein by PCR. With this method any protein sequence can be easily tagged. The techniques include three steps of DNA restriction, ligation and PCR. We could obtain a DNA construct containing SUMO-1 gene with His6 tag sequence with high efficiency by the next day.

The cDNA of BmK IT-AP, an excitatory insect toxin from the scorpion Buthus martensi Karsch that has an analgesic effect on mammalian cells, was expressed in E. coli in the form of an inclusion body. Following denaturation and reduction, the recombinant protein was renatured and purified by liquid chromatography. The authenticity of the recombinant product was confirmed by bioassay and its electrophysiological effect on ins Read More

Renaturation of horseradish peroxidase from guainidine hydrochloride has been studied. Although refolding of the secondary structure was complete, only partial heme incorporation and recovery of enzymatic activity were observed. Heme capturing became less efficient at lower peroxidase concentrations: the refolding yield decreased from 60% at 1 μM to 10% at 0.1 μM concentration of the protein. Probing with Read More

Gene 17 product (gp17) of the Pseudomonas aeruginosa-infecting bacteriophage phiKMV shows in silico similarity to T7 DNA ligase. In a semi-quantitative activity assay, it is shown that gp17 is a functional, ATP-dependent DNA ligase, in spite of some structural differences related to DNA-binding properties). Enzymatic activity of His6-based purified expression product was optimised (4°C at 24h for sticky end double-strand Read More

Arginine suppresses the aggregation of proteins. However, little is known about its mechanism. Here we have used HsNDK (Halobacterium salinarum nucleoside diphosphate kinase) to examine the solvent property of arginine. After exposure to 2 M arginine, HsNDK was diluted to a low salt buffer, resulting in fully active protein. Since unfolded HsNDK cannot refold in such low salt buffer, the observed activity indicates th Read More

Optimization of protein crystal formation is often a necessary step leading to diffraction-quality crystals to enable collection of a full X-ray data set. Typical protein crystal optimization involves screening different components, e.g., pH, precipitants, and additives of the precipitant solution. Here we present an example using an inhibitory antibody of urokinase plasminogen activator receptor (uPAR) where such procedures did not Read More

Vasostatin has previously been expressed in fused form or in inclusion body form in Escherichia coli. Here the protein was expressed in soluble non-fusion form in BL21(DE3)pLysS by IPTG induction. The expression level of vasostatin was about 15% of the total cellular protein. The expressed vasostatin was purified and its biological activity was investigated by an endothelial cell proliferation assay.

B. subtilis YjbK is a protein with 190 residues of uncharacterized function, it has been annotated by Pfam database as a member of adenylate cyclase family (EC: 4.6.1.1). In order to identify its exact function via structural studies, yjbK gene was amplified from B. subtilis genomic DNA and cloned into expression vector pET21-DEST. The protein was expressed in a soluble form in E. coli and purified to homogeneity. YjbK was Read More

The Smith-Waterman (SW) algorithm is a typical technique for local sequence alignment in computational biology. However, the SW algorithm does not consider the local behaviours of the amino acids, which may result in loss of some useful information. Inspired by the success of Markov Edit Distance (MED) method, this paper therefore proposes a novel Markov pairwise protein sequence alignment (MPPSA) method that takes Read More

A new pro-carboxypeptidase (pCPB) gene was cloned by RT-PCR from SD rat pancreas and its overexpression in Escherichia coli resulted in the formation of inclusion bodies (IBs). The IBs of pCPB were solubilized in 8 M urea and successively refolded by urea gradient gel filtration. Subsequently, the renatured pCPB was digested by trypsin. Recombinant active CPB was obtained by passing through DEAE-FF ion exch Read More

Vertebrate photoreceptor outer segment (OS) morphogenesis requires peripherin/rds (P/rds). We have characterized this protein's C-terminus and present evidence that suggests it is intrinsically disordered. We propose that structural flexibility may underlie the multifunctionality proposed for this domain previously. The extremely short Ctermini present in other tetraspanin family members suggest that intrinsic disorder may al Read More

The conformational stability of calreticulin was investigated. Apparent unfolding temperatures (Tm) increased from 31°C at pH 5 to 51°C at pH 9, but electrophoretic analysis revealed that calreticulin oligomerized instead of unfolding. Structural analyses showed that the single C-terminal α-helix was of major importance to the conformational stability of calreticulin.

A β-glucuronidase was purified from Pomacea sp. eggs by ammonium sulfate fractionation, DEAE-BioGel and Heparin-Sepharose chromatographies. This enzyme showed a Mr 180 kDa, with subunits of 90 kDa. The kinetic parameters were: pH 4.0, temperature 60°C, Km 2.7 x 10-6 and Vmax 15.3 μM/h, activator Mg+2, and inhibitor: lactone of D-saccharic acid. β-glucuronidase is an exoglucuronidase involved in Read More

Previously obtained Pic-BZA is a potent anticonvulsant with low neurotoxicity, but its half-time of action is only about 15 min. In search for equally effective anticonvulsants but with a longer time of action fourteen Pic-BZA analogs were obtained. The compounds were evaluated in the Anticonvulsant Screening Project (ASP) of Antiepileptic Drug Development Program (ADDP) of NIH. Picolinic acid 2-fluorobenzylamide (Pi Read More

An antitumour lectin named AAL has been purified from the fruiting body of edible mushroom Agrocybe aegerita. In addition to having a distinct bioactivity, AAL shows strong inhibition effects on human and mouse tumour cells. It has been shown that AAL exerts its antitumour effects via apoptosis-induction. AAL and AAL-lactose complex have been crystallized and their diffraction data were collected with resolution of 2.6 Read More

Schistosoma japonicum glutathione S-transferase (SjGST) is a common fusion tag in recombinant protein production, and its 3-dimensional structure has been studied in the context of drug design. We have determined the crystal structure of non-fused SjGST complexed with glutathione, and compare it to complexes between glutathione and SjGST fusion proteins.

Eukaryotic translation initiation factor 5A (eIF-5A) is universally found in all eukaryotic cells. It is the only protein in nature known to contain the unusual amino acid hypusine, a post-translationally modified lysine. Recombinant human eIF-5A was crystallized by the hanging-drop vapor diffusion method. Crystals were grown at 291K using (NH4)2SO4 as precipitant. Diffraction data were obtained to a resolution of 2.7Å fro Read More

B. subtilis dihydroorotase is an important enzyme in de novo pyrimidine biosynthesis pathway and encoded by pyrC gene in pyr operon. pyrC was amplified from B. subtilis genomic DNA and cloned into expression vector pET21- DEST. Dihydroorotase was expressed soluble form in E. coli and purified. The protein was crystallized and diffracted to 2.2 Å. The crystal belongs to P212121 space-group, with unit cell parameters a=4 Read More