Expression of deuterium-isotope-labelled protein in the yeast Pichia pastoris for NMR studies

Deuterium isotope labelling is important for NMR studies of large proteins and complexes. Many eukaryotic proteins are difficult to express in bacteria, but can be efficiently produced in the methylotrophic yeast Pichia pastoris. In order to facilitate NMR studies of the malaria parasite merozoite surface protein-1 (MSP1) complex and its interactions with antibodies, we have investigated production of the MSP1-19 protein in P. pastoris grown in deuterated media. The resulting deuteration patterns were analyzed by NMR and mass spectrometry. We have compared growth characteristics and levels of heterologous protein expression in cells adapted to growth in deuterated media (95% D₂O), compared with expression in non-adapted cells. We have also compared the relative deuteration levels and the distribution pattern of residual protiation in protein from cells grown either in 95% D₂O medium with protiated methanol as carbon source, or in 95% D₂O medium containing deuterated methanol. A high level of uniform C deuteration was demonstrated, and the consequent reduction of backbone amide signal linewidths in [¹H/¹⁵N]-correlation experiments was measured. Residual protiation at different positions in various amino acid residues, including the distribution of methyl isotopomers, was also investigated. The deuteration procedures examined here should facilitate economical expression of ²H/¹³C/¹⁵N-labelled protein samples for NMR studies of the structure and interactions of large proteins and protein complexes.     

Oxygen-limited fed-batch process: an alternative control for Pichia pastoris recombinant protein processes

An oxygen-limited fed-batch technique (OLFB) was compared to traditional methanol-limited fed-batch technique (MLFB) for the production of recombinant Thai Rosewood β-glucosidase with Pichia pastoris. The degree of energy limitation, expressed as the relative rate of respiration (q _O/q _O,max), was kept similar in both the types of processes. Due to the higher driving force for oxygen transfer in the OLFB, the oxygen and methanol consumption rates were about 40% higher in the OLFB. The obligate aerobe P. pastoris responded to the severe oxygen limitation mainly by increased maintenance demand, measured as increased carbon dioxide production per methanol, but still somewhat higher cell density (5%) and higher product concentrations (16%) were obtained. The viability was similar, about 90–95%, in both process types, but the amount of total proteins released in the medium was much less in the OLFB processes resulting in substantially higher (64%) specific enzyme purity for input to the downstream processing.     

Multiple gene copy number enhances insulin precursor secretion in the yeast Pichia pastoris

We have found a direct relationship between protein production in Pichia pastoris and the number of introduced synthetic genes of miniproinsulin (MPI), fused to the Saccharomyces cerevisiae pre-pro alpha factor used as secretion signal, and inserted between the alcohol oxidase 1 (AOX1) promoter and terminator sequences. Two consecutive approaches were followed to increase the number of integrated cassettes: the head-to-tail expression cassette multimerization procedure and re-transformation with a dominant selection marker. This increased expression from 19 to 250 mg l^–1 when about 11 copies have been integrated. Further, the correct position of one of the disulphide bridges of the purified molecule was verified by digestion with Glu-C endoprotease, followed by mass spectrometry of the isolated fragments.     

Molecular characterization and expression of a cDNA encoding fructan:fructan 6G-fructosyltransferase from asparagus (Asparagus officinalis)

* Fructan:fructan 6G-fructosyltransferase (6G-FFT) catalyses a transfructosylation from fructooligosaccharides to C6 of the glucose residue of sucrose or fructooligosacchrides. In asparagus (Asparagus officinalis), 6G-FFT is important for the synthesis of inulin neoseries fructan. Here, we report the isolation and functional analysis of the gene encoding asparagus 6G-FFT. * A cDNA clone was isolated from asparagus cDNA library. Recombinant protein was produced by expression system of Pichia pastoris. To measure enzymatic activity, recombinant protein was incubated with sucrose, 1-kestose, 1-kestose and sucrose, or neokestose. The reaction products were detected by high performance anion-exchange chromatography. * The deduced amino acid sequence of isolated cDNA was similar to that of fructosyltransferases and vacuolar type invertases from plants. Recombinant protein mainly produced inulin neoseries fructan, such as 1F, 6G-di-beta-D-fructofuranosylsucrose and neokestose. * Recombinant protein demonstrates 6G-FFT activity, and slight fructan:fructan 1-fructosyltransferase (1-FFT) activity. The ratio of 6G-FFT activity to 1-FFT activity was calculated to be 13. The characteristics of the recombinant protein closely resemble those of the 6G-FFT from asparagus roots, except for a difference in accompanying 1-FFT activity.     

泥鳅抗菌肽在毕赤酵母SMD1168中的高效表达及活性检测

【背景】泥鳅抗菌肽Misgurin是泥鳅非特异性免疫防御系统的重要组成部分,具有广谱和较强的抗菌能力,所以获得大量抗菌肽是很有必要的。【目的】为了实现高效表达泥鳅抗菌肽Misgurin。【方法】将泥鳅抗菌肽的目的基因与p PIC9K表达载体连接,构建重组表达质粒p PIC9K-misgurin,Sal I酶切线性化,再通过电击法将其整合到毕赤酵母SMD1168染色体上。在MD固体培养基挑选阳性克隆子到MD液体培养基中,30°C、200 r/min摇瓶培养96 h,转接到BMMY液体培养基进行诱导表达,每隔24 h加入5%的甲醇。通过比较抑制大肠杆菌和金黄色葡萄球菌抑菌圈直径的大小,筛选出活性较高的菌株p PIC9K-misgurin-22。将该菌株在100 L的发酵罐中诱导表达,48 h后进行活性检测。【结果】经Tricine-SDS-PAGE蛋白胶检测和质谱分析鉴定,p PIC9K-misgurin-22菌株诱导表达的活性物质为抗菌肽Misgurin。发酵罐发酵48 h相对于摇瓶发酵48 h,抗大肠杆菌的生物效价提高了1.47倍,抗金黄色葡萄球菌的生物效价提高了1.43倍;抗菌肽Misgurin对鲍曼不动杆菌、沙门氏菌有较弱的抗菌活性,对致病菌产气荚膜梭菌和益生菌如枯草芽孢杆菌、粪肠球菌、乳酸菌没有抗菌活性,无明显溶血活性;当温度达到90°C时发酵液的抗菌活性明显减弱,调发酵液的p H值在1.0-12.0之间都有抗菌活性;加入胰蛋白酶、胃蛋白酶、蛋白酶K后抗菌活性减弱。【结论】获得了一株在毕赤酵母中表达量较高、有工业化生产潜力的产抗菌肽Misgurin的菌株。     

Location of tyrosine phenol-lyase in some Gram-negative bacteria

Abstract From various habitats (plant material, fruits, soil), yeasts belonging to the species of Pichia kluyveri and Hanseniaspora uvarum were isolated that showed killer activity. According to the activity spectrum against other yeasts these strains belonged to 11 different groups that were distinguishable from the killer strains K₁-K₁₀. The isoelectric points of the killer proteins were in the range of pH 3.5–3.9, the activity optimum was observed at pH 4.2–4.6. Above pH 5 and above a temperature of 25–35°C the killer proteins were inactivated.     

Effect of aerobiosis on fermentation and key enzyme levels during growth of Pichia stipitis,Candida shehatae and Candida tenuis on d-xylose

The relationship between the degree of aerobiosis, xylitol production and the initial two key enzymes of d-xylose metabolism were investigated in the yeasts Pichia stipitis, Candida shehatae and C. tenuis. Anoxic conditions severely curtailed growth and retarded ethanol productivity. This, together with the inverse relationship between xylitol accumulation and aeration level, suggested a degree of redox imbalance. The ratios of NADH- to NADPH-linked xylose reductase were similar in all three yeasts and essentially independent of the degree of aerobiosis, and thus did not correlate with their differing capacities for ethanol production, xylitol accumulation or growth under the different conditions of aerobiosis. Under anoxic conditions the enzyme activity of Pichia stipitis decreased significantly, which possibly contributed to its weaker anoxic fermentation of xylose compared to C. shehatae.     

Determination of kinetic parameters of fermentation processes by a continuous unsteady-state method: application to the alcoholic fermentation of D-xylose by Pichia stipitis

A quick technique for determination of kinetic parameters of fermentation processes is proposed and applied to the transformation of D-xylose into ethanol by Pichia stipitis. The commonly used method to evaluate these parameters is based on achieving several steady states. In the proposed procedure, mu(m) and K(s) can be determined from only one steady state, by provoking a disturbance over it, after allowing the system to return to the original conditions. The main difference between the steady and unsteady state methods is the required fermentation time; while the former method lasted 350 h, the latter required a period 25 times lower. Kinetic and stoichiometric parameters were determined with both methods under anoxic and limited oxygen concentration conditions. Results from the two methods were compared, giving only 2% and 4.5% differences in the values of K(s) and mu(m) and a little over 4% for mu(m) were the deviations under the latter ones. (c) 1993 Wiley & Sons, Inc.     

Killer toxin secretion through the cell wall of the yeastPichia anomala

A secreted killer toxin was detected through the cell wall ofPichia anomala cells by ultrastructural immunodetection with a specific monoclonal antibody (MAb KT4). MAb KT4 was successively detected by colloidal gold labeled streptavidin and biotinylated anti-mouse F(ab')₂ antibodies. The antigenic determinants of the toxin were localized throughout the cytoplasm and the cell wall of killer yeast cells. The Lowicryl K4M-immunogold method gave very satisfactory results and showed that the killer toxin was somewhat concentrated in the yeast cell wall layers before being exported into the medium. In agreement with previous reports, the binding of MAb KT4 suggested that theP. anomala killer toxin secretion did not result from a homogeneous diffusion across the yeast cell wall.Abbreviations G15 gold particles of 15 nm - IEM immunoelectron microscopy - IFA immunofluorescence assay - MAb monoclonal antibody - PBS phosphate buffered saline - SAM/F(ab)₂ sheep antibodies anti-mouse F(ab)₂ - SBB Sabouraud buffered broth     

Recombinant Pichia pastoris overexpressing bioactive phytase

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