Yeast-based system for in vivo evaluation of alleles of the anthocyanin production pathway.

Plants produce anthocyanins to incite the pollination and seed dispersion performed by pigment-attracted animals. These natural blue-to-red-coloured pigments can be used as food colourants and antioxidants. For this purpose, microbial bioproduction of anthocyanins has become of industrial interest in recent years. 20 new alleles of anthocyanin production pathway genes were extracted and characterised for protein expression level and stability using a developed single-PCR product gene-entry system for tagged protein synthesis in yeast S. cerevisiae. Enzymatic activities of these proteins in the episomally complemented in vivo systems were compared by HPLC-MS analysis. Results show that the codon optimisation of the anthocyanin pathway genes is not essential for the effective heterologous expression in yeast. Elevating the cellular abundance of CHS and F3H enzymes can increase anthocyanidin production from supplemented precursors. New alleles VmF3Hv1 and VuCHS were shown to have the best performance in the analysed system. System complementation with flavonoid 3',5'-hydroxylase substantially increases total anthocyanidin production. The described single-entry yeast episomal complementation system is a convenient and rapid tool for the complex evaluation of new alleles in vivo.

Purification and characterization of the recombinant human prostaglandin H synthase-2 expressed in Pichia pastoris.

Prostaglandin H synthase-1 and -2 (PGHS-1 and PGHS-2, EC 1.14.99.1) are membrane associated glycoproteins that catalyze the first two steps in prostaglandin synthesis. As the enzymes play an important regulatory role in several physiological and pathophysiological processes, recombinant PGHS isoforms are widely used in biomedical research. In the present study, we expressed human PGHS-2 (hPGHS-2) with and without a six histidine sequence tag (His(6) tag) near the amino- or carboxy-terminus of the protein in the Pichia pastoris (P. pastoris) expression system using native or yeast signal sequences. The recombinant His(6) tagged hPGHS-2 was purified using Ni-affinity and anion exchange chromatography, whereas the purification of the C-terminally His(6) tagged hPGHS-2 was more efficient. K(m), k(cat) and IC(50) values were determined to characterize the protein. The data obtained indicate that both the N- and C-terminally His(6) tagged hPGHS-2 are functional and the catalytic properties of the recombinant protein and the enzyme produced in other expression systems are comparable. As the yeast culture is easy to handle, the P. pastoris system could serve as an alternative to the most commonly used baculovirus-insect cell expression system for the production of the recombinant PGHS-2.

Calorimetric approach for comparison of Angiopoietin-like protein 4 with other pancreatic lipase inhibitors.

Pancreatic lipase (PNLIP) is a digestive enzyme that is a potential drug target for the treatment of obesity. A better understanding of its regulation mechanisms would facilitate the development of new therapeutics. Recent studies indicate that intestinal lipolysis by PNLIP is reduced by Angiopoietin-like protein 4 (ANGPTL4), whose N-terminal domain (nANGPTL4) is a known inactivator of lipoprotein lipase (LPL) in blood circulation and adipocytes. To elucidate the mechanism of PNLIP inhibition by ANGPTL4, we developed a novel approach, using isothermal titration calorimetry (ITC). The obtained results were compared with those of well-described inhibitors of PNLIP - ε-polylysine (EPL), (-)-epigallocatechin-3-gallate (EGCG) and tetrahydrolipstatin. We demonstrate that ITC allows to investigate PNLIP inhibition mechanisms in complex substrate emulsions and that the ITC-based assay is highly sensitive - the lowest concentration for quantification of PNLIP is 1.5 pM. Combining ITC with surface plasmon resonance and fluorescence measurements, we present evidence that ANGPTL4 is a lipid-binding protein that influences PNLIP activity through interactions with components of substrate emulsions (bile salts, phospholipids and triglycerides), and this promotes the aggregation of triglyceride emulsions similarly to the PNLIP inhibitors EPL and EGCG. In the absence of substrate emulsion, unlike in the case of LPL, ANGPTL4 did not induce the inactivation of PNLIP. Our data also prove that due to various interactions with components of substrate systems, the effect of a PNLIP inhibitor depends on whether its effect is measured in a complex substrate emulsion or in a simple substrate system.

Enhanced expression of human prostaglandin H synthase-2 in the yeast Pichia pastoris and removal of the C-terminal tag with bovine carboxypeptidase A.

Vertebrate prostaglandin H synthases (PGHSs) are membrane-bound disulphide-containing hemoglycoproteins. Therefore, eukaryotic expression systems are required for the production of recombinant PGHSs. Recently we announced the expression of human PGHS-2 (hPGHS-2) in the yeast Pichia pastoris. Here we report improved production of hPGHS-2 in P. pastoris and a convenient method for the purification and de-tagging of the protein. An affinity tag comprised of a proline, a glycine and eight histidines was introduced into the C-terminal end of hPGHS-2. The tagged hPGHS-2 was expressed intracellularly in P. pastoris under the control of a constitutive or methanol-inducible promoter. Compared to constitutive expression, methanol-induced expression yielded approximately four times more protein. The analysis of high and low gene copy number recombinants revealed a positive correlation between the gene copy number and the expression level of hPGHS-2. The recombinant hPGHS-2 was purified using immobilised metal ion affinity chromatography. A novel elution method, treatment of the affinity resin with bovine carboxypeptidase A, was employed. The yield of pure de-tagged hPGHS-2 from 1l of yeast culture was approximately 3mg. The protein purification process with simultaneous removal of the C-terminal polyhistidine tag could be easily applied for the affinity purification of other proteins.

N-glycosylation site occupancy in human prostaglandin H synthases expressed in Pichia pastoris.

Prostaglandin H synthases (PGHSs) are N-glycosylated membrane proteins that catalyse the committed step in prostaglandin synthesis. Unlike PGHS-2, the production of recombinant PGHS-1 in non-mammalian expression systems is complicated. The majority of the heterologous enzyme is inactive due to misfolding. Correct N-glycosylation is proposed to be obligatory for proper folding of mammalian PGHSs. In this study, human PGHS-1 and -2 (hPGHS-1 and -2) were expressed in the yeast Pichia pastoris. Recombinant hPGHS-2 was catalytically active, whereas hPGHS-1 was inactive. Accumulation of non-glycosylated hPGHSs was not observed in the crude lysate of the yeast cells. The N-glycosylation patterns of the purified recombinant proteins were characterised using nano-LC/MS/MS. The isoforms exhibited similar N-glycosylation site occupancy. The results indicate that there are more complex grounds for the inactivity of the recombinant hPGHS-1 produced in yeast.