Production of membrane proteins in yeast

Background Yeast is an important and versatile organism for studying membrane proteins. It is easy to cultivate and can perform higher eukaryote-like post-translational modifications. S. cerevisiae has a fully-sequenced genome and there are several collections of deletion strains available, whilst P. pastoris can produce very high cell densities (230 g/l). Results We have used both S. cerevisiae and P. pastoris to over-produce the following His6 and His10 carboxyl terminal fused membrane proteins. CD81 – 26 kDa tetraspanin protein (TAPA-1) that may play an important role in the regulation of lymphoma cell growth and may also act as the viral receptor for Hepatitis C-Virus. CD82 – 30 kDa tetraspanin protein that associates with CD4 or CD8 cells and delivers co-stimulatory signals for the TCR/CD3 pathway. MC4R – 37 kDa seven transmembrane G-protein coupled receptor, present on neurons in the hypothalamus region of the brain and predicted to have a role in the feast or fast signalling pathway. Adt2p – 34 kDa six transmembrane protein that catalyses the exchange of ADP and ATP across the yeast mitochondrial inner membrane. Conclusion We show that yeasts are flexible production organisms for a range of different membrane proteins. The yields are such that future structure-activity relationship studies can be initiated via reconstitution, crystallization for X-ray diffraction or NMR experiments.

A functional chimeric DNA primase: the Cys4 zinc-binding domain of bacteriophage T3 primase fused to the helicase of bacteriophage T7

Two colinear bacteriophage T7 gene 4 proteins provide helicase and primase functions in vivo. T7 primase differs from T7 helicase by an additional 63 residues at the amino terminus. This terminal domain contains a zinc-binding motif which mediates an interaction with the basic primase recognition sequence 3'-CTG-5'. We have generated a chimeric primase in which the 81 amino-terminal residues are derived from the primase of phage T3 and the 484 carboxyl-terminal residues are those of phage T7 helicase. The amino-terminal domain of T3 primase is 50% homologous with that of T7 primase. The resulting T3/T7 chimeric protein is a functional primase in vivo. While the primase activity of the purified protein is about one-third that of T7 primase, the recognition sites used and the oligoribonucleotides synthesized from these sites are identical. We conclude that the residues responsible for the interaction with the sequence 3'-CTG-5' are conserved between the chimeric and T7 proteins.

Nitric oxide in the rat gastrointestinal tract

This study concerns the nature of nitric oxide synthase (NOS) and the role of nitric oxide (NO) in the rat gastrointestinal tract. The major objectives were (i) to characterise NOS isoforms in the gastric glandular mucosa, (ii) to localise NOS isoforms in the rat gastric glandular mucosa, (iii) to investigate the role of NO in carbachol-stimulated gastric mucus secretion, (iv) to investigate the nature of NOS and small intestine. Immunoblotting was performed using polyclonal antisera raised against two peptides found in the rat brain NOS sequence and commercial monoclonal antibodies directed against neuronal and endothelial isoforms of NOS. A160kDa band was detected in brain and gastric mucosal samples with antibodies and antisera directed against neuronal NOS sequences, and a 140kDa band was detected in gastric mucosal samples using an anti-endothelial NOS antibody. An intense 160kDa neuronal NOS band was detected in a high-density fraction of gastric mucosal cells separated on a Percoll gradient. Detection of neuronal NOS by a carboxyl-terminal antiserum in samples of brain, but not of gastric mucosa, could be blocked by the peptide (20g/ml) against which the antibody was raised. After affinity purification, recognition of gastric mucosal NOS was blocked by peptide. Particulate neuronal NOS was found in the brain by immunoblotting while 94% of gastric mucosal enzyme was soluble. Gastric mucosal endothelial NOS was 95% particulate. 95% of NOS activity in the gastric mucosa was due to neuronal NOS. Paraformaldehyde- and acetone-fixed gastric mucosal sections were subject to immunocytochemistry using the above antibodies. Neuronal NOS was localised to the surface mucosal epithelial cells while endothelial NOS was associated with microvessels at the base of the mucosa and to larger vessels in the submucosa. Intragastric administration of carbachol or 16, 16-dimethyl prostaglandin E2 increased the thickness of the rat gastric mucus layer. The NOS inhibitor NG-nitro-L-arginine methyl ester dose-dependently, and selectively, prevented the stimulatory effect of carbachol. Ca2+-independent NOS activity in rat ileal, jejunal and colonic muscle was increased after LPS induction. Ca2+-dependent activity was not affected. Distribution of inducible NOS protein paralleled Ca2+ -independent activity. LPS treatment did not affect the content of neuronal NOS in colonic muscle.

Crystal structure of [{Cu(H2O)(en)2}{Cu(en)2}Re6Te8(CN)6]·3H2O

The reaction of Cs4[Re6Te8(CN)6]·2H2O with Cu(en)2Cl2 in water affords crystals of a cluster complex [{Cu(H2O)(en) 2}{Cu(en)2}Re6Te8(CN)6]·3H2O. The structure of the compound is determined by single crystal X-ray diffraction (a = 10.8082(4) Å, b = 16.5404(6) Å, c = 24.6480(7) Å, β = 92.696(1)°, V = 4401.5(3) Å3, Z = 4, space group P21/n, R 1 = 0.0331, wR 2 (all data) = 0.0652). In the complex, cluster [Re6Te8(CN)6]4- anions are linked by Cu2+ cations into zigzag chains through cyanide bridges. The coordination environment of the copper cations is complemented by ethylenediamine molecules. Each of the cluster anions is additionally coordinated by a terminal fragment {Cu(H2O)(en)2}. © 2014 Pleiades Publishing, Ltd.