Structural insights into the human RyR2 N-terminal region involved in cardiac arrhythmias.

Human ryanodine receptor 2 (hRyR2) mediates calcium release from the sarcoplasmic reticulum, enabling cardiomyocyte contraction. The N-terminal region of hRyR2 (amino acids 1-606) is the target of >30 arrhythmogenic mutations and contains a binding site for phosphoprotein phosphatase 1. Here, the solution and crystal structures determined under near-physiological conditions, as well as a homology model of the hRyR2 N-terminal region, are presented.

Human cardiac ryanodine receptor: preparation, crystallization and preliminary X-ray ANALysis of the N-terminal region.

Human ryanodine receptor 2 (hRyR2) is a calcium ion channel present in the membrane of the sarcoplasmic reticulum of cardiac myocytes that mediates release of calcium ions from the sarcoplasmic reticulum stores during excitation- contraction coupling. Disease-causing mutations of hRyR2 are clustered into N-terminal (amino acids 1-600), central (amino acids 2100-2500) and C-terminal (amino acids 3900-5000) regions. These regions are believed to be involved in regulation of channel gating.

Gene sequence, bioinformatics and enzymatic characterization of alpha-amylase from Saccharomycopsis fibuligera KZ.

A fragment coding for a putative extracellular alpha-amylase, from the genomic library of the yeast Saccharomycopsis fibuligera KZ, has been subcloned into yeast expression vector pVT100L and sequenced. The nucleotide sequence revealed an ORF of 1,485 bp coding for a 494 amino acid residues long protein with 99% identity to the alpha-amylase Sfamy from S. fibuligera HUT 7212.

Bioinformatic mapping and production of recombinant N-terminal domains of human cardiac ryanodine receptor 2.

We report the domain analysis of the N-terminal region (residues 1-759) of the human cardiac ryanodine receptor (RyR2) that encompasses one of the discrete RyR2 mutation clusters associated with catecholaminergic polymorphic ventricular tachycardia (CPVT1) and arrhythmogenic right ventricular dysplasia (ARVD2). Our strategy utilizes a bioinformatics approach complemented by protein expression, solubility analysis and limited proteolytic digestion. Based on the bioinformatics analysis, we designed a series of specific RyR2 N-terminal fragments for cloning and overexpression in Escherichia coli.

Antibody response to the 45 kDa Candida albicans antigen in an animal model and potential role of the antigen in adherence.

The Candida antigen CR3-RP (complement receptor 3-related protein) is supposed to be a 'mimicry' protein because of its ability to bind antibody directed against the alpha subunit of the mammalian CR3 (CD11b/CD18). This study aimed to (i) investigate the specific humoral isotypic response to immunization with CR3-RP in vivo in a rabbit animal model, and (ii) determine the role of CR3-RP in the adherence of Candida albicans in vitro using the model systems of buccal epithelial cells (BECs) and biofilm formation. The synthetic C.

Structure of the complex of a yeast glucoamylase with acarbose reveals the presence of a raw starch binding site on the catalytic domain.

Most glucoamylases (alpha-1,4-D-glucan glucohydrolase, EC 3.2.1.

Expression and purification of human antimicrobial peptide, dermcidin, in Escherichia coli.

Human dermcidin, an anionic antimicrobial peptide expressed in the pons of the brain and the sweat glands, displays antimicrobial activity against pathogenic microorganisms such as Staphylococcus aureus and Candida albicans. Here, we describe the recombinant production of a 48 amino acid dermcidin variant with C-terminal homoserine lactone (DCD-1Hsl). Dermcidin coding sequence was cloned downstream of a 125 amino acid ketosteroid isomerase gene and upstream of a His6Tag sequence in pET-31b(+) vector and transformed into Escherichia coli.

Cloning and expression of a gene for an alpha-glucosidase from Saccharomycopsis fibuligera homologous to family GH31 of yeast glucoamylases.

Cloning of cDNA encoding an alpha-glucosidase from the dimorphous yeast Saccharomycopsis fibuligera and characterization of the gene product were performed. The cDNA of the putative alpha-glucosidase gene consists of 2,886 bp, which includes an open reading frame encoding a 19 amino acid signal peptide at the N-terminal end and a 944 amino acid mature protein with a predicted molecular mass of 105.4 kDa and pI value of 4.

High-level expression and purification of a recombinant hBD-1 fused to LMM protein in Escherichia coli.

In this work, we present the production of an active 43 aa recombinant human beta-defensin-1 (rhBD-1(43)) in Escherichia coli AD202 cells using specific pLMM1-rhBD-1 expression system. Unique solubility properties of the C-terminal fragment of light meromyosin (LMM) allowed us to overcome foreseeable problems with isolation procedures and toxicity caused by rhBD-1 to the host organism. As a result, the majority of fusion protein (LMM-rhBD-1(43)) was obtained in the soluble state, isolated by a low salt-high salt treatment of total cell protein.

Expression, purification, and sequence analysis of catalase-1 from the soil bacterium Comamonas terrigena N3H.

Catalases are essential components of the cellular equipment to cope with oxidative stress. We have purified and characterize herein the most abundant heme-containing catalase-1 from the soil bacterium Comamonas terrigena N3H. This oxidative stress-induced enzyme was isolated from exponential phase cells grown in the presence of peroxyacetic acid.

Effect of fast protein liquid chromatography fractionated salivary gland extracts from different ixodid tick species on interleukin-8 binding to its cell receptors.

Interleukin-8 plays a critical role in inflammatory processes. Hence generation of molecules with anti-IL-8 activity is likely to be important for successful feeding and for survival of the ticks. Anti-IL-8 activity was studied in saliva of three ixodid tick species--Dermacentor reticulatus (Fabricius, 1794), Rhipicephalus appendiculatus Neumann, 1901, and Amblyomma variegatum (Fabricius, 1794).

Molecular cloning and 3D structure prediction of the first raw-starch-degrading glucoamylase without a separate starch-binding domain.

Raw-starch-degrading glucoamylases have been known as multidomain enzymes consisting of a catalytic domain connected to a starch-binding domain (SBD) by an O-glycosylated linker region. A molecular genetics approach has been chosen to find structural differences between two related glucoamylases, raw-starch-degrading Glm and nondegrading Glu, from the yeasts Saccharomycopsis fibuligera IFO 0111 and HUT 7212, respectively. We have found that Glm and Glu show a high primary (77%) and tertiary structure similarity.