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.     

Fermentation strategies for recombinant protein expression in the methylotrophic yeastPichia pastoris

Fermentation strategies for recombinant protein production inPichia pastoris have been investigated and are reviewed here. Characteristics of the expression system, such as phenotypes and carbon utilization, are summarized. Recently reported results such as growth model establishment, application of a methanol sensor, optimization of substrate feeding strategy, DOstat controller design, mixed feed technology, and perfusion and continuous culture are discussed in detail.     

Efficient secretion of human endostatin in the yeast, Pichia pastoris

Endostatin is a 20 kDa COOH-terminal fragment of collagen XVIII that inhibits angiogenesis and tumor growth. The cDNA coding for human endostatin in human fetal liver has been cloned into the secreting expression organism Pichia pastoris, and the high level expression of human endostatin has been achieved (about 200 mg of endostatin in 1 l of culture). The recombinant human endostatin was purified to homogeneity by heparin-affinity column, and showed antiproliferative effect on rat brain micro-vascular endothelia cells.     

Expression in Pichia pastoris and immunological evaluation of a truncated dengue envelope protein

Among the Dengue virus structural proteins, the Envelope glycoprotein is the most important because of its antigenic characteristics. In this work, the E protein from Dengue-2 virus truncated at the C-terminus region was successfully expressed in Pichia pastoris. The E2trunc gene was cloned under the AOX1 promoter from P. pastoris and the signal peptide of the sucrose invertase gene from Saccharomyces cerevisiae. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of expression revealed the presence of a protein with the expected size, which was completely associated to the insoluble fraction after cellular disruption. The recombinant N-glycosylated protein reacted with two conformational antibodies against Dengue-2, indicating a proper folding of it. In addition, it was able to induce antiviral antibodies after mice immunization.     

栓菌漆酶在毕赤酵母中高效表达及重组酶的性质

栓菌420（Trametes sp. 420）漆酶基因lacD以两种方式在巴斯德毕赤酵母（Pichia pastoris）进行异源表达,产生两种重组漆酶：rLacDx（具有天然N-末端）和rLacDe（N-末端带有8个额外的氨基酸残基）。摇瓶发酵18d,rLacDx和rLacDe的产量分别为1.21×10⁵u/L、7.38×10⁴u/L ［以2,2′-连氮-3-乙苯-二噻唑-6磺酸（ABTS）为底物］。在高密度发酵条件下,rLacDx的产量增加到2.39×10⁵u/L,同时其生产周期降至7.5 d。两种重组酶对愈创木酚底物的氧化特性相似,且在50℃和pH3～10的范围内均稳定。然而,rLacDx对底物ABTS的比活力（1761u/mg）高于rLacDe （1122u/mg）,其表观K_m值(427μmol/L)低于rLacDe (604μmol/L)。     

Effects of methanol concentration on expression levels of recombinant protein in fed-batch cultures of Pichia methanolica

The methylotrophic yeast Pichia methanolica can be used to express recombinant genes at high levels under the control of the methanol-inducible alcohol oxidase (AUG1) promoter. Methanol concentrations during the induction phase directly affect cellular growth and protein yield. Various methanol concentrations controlled by an on-line monitoring and control system were investigated in mixed glucose/methanol fed-batch cultures of P. methanolica expressing the human transferrin N-lobe protein. The PMAD18 P. methanolica strain utilized is a knock-out for the chromosomal AUG1 gene locus, resulting in a slow methanol utilization phenotype. Maximum growth of 100 g of dry cell weight per liter of culture was observed in cultures grown at 1.0% (v/v) methanol concentration. Maximum recombinant gene expression was observed for cultures controlled at 0.7% (v/v) methanol concentration, resulting in maximum volumetric production of 450 mg of transferrin per liter after 72 h of elapsed fermentation time.     

Constitutive expression of human pancreatic lipase-related protein 1 in Pichia pastoris

High-level constitutive expression of the human pancreatic lipase-related protein 1 (HPLRP1) was achieved using the methylotrophic yeast Pichia pastoris. The HPLRP1 cDNA, including its original leader sequence, was subcloned into the pGAPZB vector and further integrated into the genome of P. pastoris X-33 under the control of the glyceraldehyde 3-phosphate dehydrogenase (GAP) constitutive promoter. A major protein with a molecular mass of 50 kDa was found to be secreted into the culture medium and was identified using anti-HPLRP1 polyclonal antibodies as HPLRP1 recombinant protein. The level of expression reached 100-120 mg of HPLRP1 per liter of culture medium after 40 h, as attested by specific and quantitative enzyme-linked immunosorbent assay. A single cation-exchange chromatography sufficed to obtain a highly purified recombinant HPLRP1 after direct batch adsorption onto S-Sepharose of the HPLRP1 present in the culture medium, at pH 5.5. N-terminal sequencing and mass spectrometry analysis were carried out to monitor the production of the mature protein and to confirm that its signal peptide was properly processed.     

Increasing the cell viability and heterologous protein expression of Pichia pastoris mutant deficient in PMR1 gene by culture condition optimization

In this study, we assessed the potential of PMR1-disrupted Pichia pastoris (Pppmr1) expressing human serum albumin and interferon alpha2b fusion protein (HSA-IFN-alpha2b) in large-scale fermentation. The high osmotic pressure of standard basal salts medium (BSM) was detrimental to the growth and viability of Pppmr1. HSA-IFN-alpha2b was secreted into a supernatant with a concentration of up to 112 mg/L after 20 h of induction and then began to decline. In vitro stability tests indicated that the disappearance of HSA-IFN-alpha2b was ascribed to proteolytic degradation. Decreasing the salt concentration of BSM medium to one quarter of the original formula improved the growth and viability of Pppmr1. As a result of reduced cell lysis and protease release, HSA-IFN-alpha2b was stable in the supernatant, which enabled a longer production phase (30 h) and a higher expression level (215 mg/L). Lowering the culture temperature to 20°C increased the cell viability during carbon source transition and alleviated the oxygen and methanol limitation, which extended the production phase to 40 h and increased the expression level to 680 mg/L. The addition of 2% Soytone prolonged the production phase to 60 h and increased the expression level to 1,260 mg/L, which was more than tenfold higher than that of Pppmr1 cultured under the conditions recommended by Invitrogen.     

Functional expression of keratinase (kerA) gene from Bacillus licheniformis in Pichia pastoris

A 1.2 kb DNA fragment coding for the pro-peptide and mature keratinase from Bacillus licheniformis PWD-1 (kerA) was cloned into vectors pPICZA and pGAPZA for extracellular expression in the methylotrophic yeast, Pichia pastoris. Recombinant keratinase was secreted by the pPICZA-kerA transformants 24 h after methanol induction of shake-flask cultures, and reached a final yield of 124 mg l^–1 (285 U ml^–1) 144 h after the induction. The recombinant keratinase was glycosylated ( 39 kDa), and was optimal between pH 8.5–9.5 and between 55°C –60°C using azokeratin as substrate. The enzyme degraded bovine serum albumin, collagen, and soy protein concentrate. In conclusion, P. pastoris can be used as an efficient host to express keratinase for nutritional and environmental applications.     

Modeling of mini-proinsulin production in Pichia pastoris using the AOX promoter

An unstructured model based on mass balance equations for a recombinant methylotrophic yeast Pichia pastoris Mut^S (Methanol Utilization Slow) strain expressing the mini-proinsulin (MPI), was successfully established in quasi-steady state fed-batch fermentations with varying total quantity of biomass in a 7 l fermenter. The model describes the relationships between the total biomass and induction time, both in the batch and fed-batch phases. In addition, good correlations were obtained when the total quantity of MPI was correlated with the total biomass, demonstrating that the product of interest is associated with growth in the methanol phase. The parameters of the fermentation model obtained are similar to those reported by other researchers.     

High level expression of recombinant Mycobacterium tuberculosis culture filtrate protein CFP32 in Pichia pastoris

Difficulty in obtaining large quantities of Mycobacterium tuberculosis (MTB) proteins remains a major obstacle in the development of subunit vaccines and diagnostic reagents for tuberculosis. A major reason is because Escherichia coli has not proven to be an optimal host for the expression of MTB genes. In this article, we used the yeast Pichia pastoris to express high levels of CFP32, a culture filtrate protein restricted to the MTB complex and a potential target antigen for serodiagnosis of tuberculosis in patients. Using shaker flasks, we generated a P. pastoris clone expressing CFP32 as a secreted protein fused to the myc-(His)₆ tag, at a yield of 0.5 g of purified protein per liter of culture. Recombinant CFP32 (rCFP32) produced in P. pastoris has a molecular weight of 35 kDa, which is slightly higher than that of the native protein We identified putative acylation and glycosylation sites in the CFP32 amino acid sequence that suggested post-translational modifications may contribute to the size difference. The NH₂-terminal peptide sequencing of rCFP32 showed that the signal peptide alpha factor is correctly excised. In addition, rCFP32 reacted with the sera of patients with tuberculosis. These data are the first to show that P. pastoris is a suitable host for high-yield production of good quality mycobacterium antigens, and especially culture filtrate proteins that have vaccine and diagnostic potential.     

Optimal conditions for the expression of a single-chain antibody (scFv) gene in Pichia pastoris

A Pichia pastoris system was used to express a single-chain antibody (scFv) targeted against Mamestra configurata (bertha armyworm) serpins. To improve scFv production we examined parameters such as proteinase activity, temperature, cell density, osmotic stress, medium composition, pH, and reiterative induction. P. pastoris was found to express several proteases; however, adjustment of medium pH to limit their activity did not correlate with increased scFv recovery. Induction medium pH values of 6.5-8.0 were most conducive to scFv production, despite significant differences in cell growth rates. Increasing inoculum density limited growth potential but gave rise to higher levels of scFv production. Three factors, medium composition, pre-induction osmotic stress, and temperature, had the greatest effects on protein production. Supplementation of the induction medium with arganine, casamino acids, or EDTA increased scFv production several fold, as did cultivation under osmotic stress conditions during pre-induction biomass accumulation. Incubation at 15 versus 30 degrees C extended the period whereby cells were capable of producing scFv from 1 to 7 days. Under optimal conditions, yeast cultures yielded 25 mg/L of functional scFv and could be subject to five reiterative inductions.     

Expression of recombinant actin 5C from Drosophila in the methylotrophyc yeast Pichia pastoris

A system of expression for the foreign actin gene in yeast cells Pichia pastoris has been developed. As a target protein, the Drosophila cytoplasmic actin 5C, which has 90% homology to the β-actin of higher eukaryotes, was used. In the present work, in order to develop conditions for biosynthesis of the target protein in yeast cells and a purification procedure for the recombinant protein, a GFP-actin fusion protein containing green fluorescent protein (GFP) as a fusion tag was expressed and purified. The size and survival of P. pastoris cells producing recombinant protein were characterized and shown to depend on the accumulation of recombinant actin. The purified fusion protein was used to obtain a polyclonal antibody necessary for testing for recombinant actin.     

Efficient expression and purification of human interferon alpha2b in the methylotrophic yeast, Pichia pastoris

The human interferon alpha2b (hIFN-alpha2b) is the most widely used member of IFNalpha family, and it exerts many biological actions including broad-spectrum antiviral effects, inhibition of tumor cell proliferation and enhancement of immune functions. Herein, the cDNA coding for hIFN-alpha2b has been cloned into the secreting expression organism Pichia pastoris, and the high level expression of hIFN-alpha2b has been achieved. SDS-PAGE and Western blotting assays of culture broth from a methanol-induced expression strain demonstrated that recombinant hIFN-alpha2b, a 18.8 kDa protein, was secreted into the culture medium. The recombinant protein was purified to greater than 95% using Source Q ion exchange and Superdex 75 size-exclusion chromatography steps. Finally, 298 mg of the protein was obtained in high purity from 1l of the supernatant and its identity to hIFN-alpha2b was confirmed by NH(2)-terminal amino acid sequence analysis. The bioassay of the recombinant protein gave a specific activity of 1.9 x 10(9)IU/mg. Our results suggest that the P. pastoris expression system can be used to produce large quantities of fully functional hIFN-alpha2b for both research and industrial purpose.     

Relative and accurate measurement of protein abundance using 15N stable isotope labeling in Arabidopsis (SILIA)

In the quantitative proteomic studies, numerous in vitro and in vivo peptide labeling strategies have been successfully applied to measure differentially regulated protein and peptide abundance. These approaches have been proven to be versatile and repeatable in biological discoveries. ¹⁵N metabolic labeling is one of these widely adopted and economical methods. However, due to the differential incorporation rates of ¹⁵N or ¹⁴N, the labeling results produce imperfectly matched isotopic envelopes between the heavy and light nitrogen-labeled peptides. In the present study, we have modified the solid Arabidopsis growth medium to standardize the ¹⁵N supply, which led to a uniform incorporation of ¹⁵N into the whole plant protein complement. The incorporation rate (97.43 ± 0.11%) of ¹⁵N into ¹⁵N-coded peptides was determined by correlating the intensities of peptide ions with the labeling efficiencies according to Gaussian distribution. The resulting actual incorporation rate (97.44%) and natural abundance of ¹⁵N/¹⁴N-coded peptides are used to re-calculate the intensities of isotopic envelopes of differentially labeled peptides, respectively. A modified ¹⁵N/¹⁴N stable isotope labeling strategy, SILIA, is assessed and the results demonstrate that this approach is able to differentiate the fold change in protein abundance down to 10%. The machine dynamic range limitation and purification step will make the precursor ion ratio deriving from the actual ratio fold change. It is suggested that the differentially mixed ¹⁵N-coded and ¹⁴N-coded plant protein samples that are used to establish the protein abundance standard curve should be prepared following a similar protein isolation protocol used to isolate the proteins to be quantitated.     

Co-expression of human protein disulfide isomerase (hPDI) enhances secretion of bovine follicle-stimulating hormone (bFSH) in Pichia pastoris

Bovine follicle-stimulating hormone (bFSH) is a pituitary gonadotropin composed of two non-covalently associated polypeptide subunits, which must be glycosylated, folded, and assembled as a heterodimer to be biologically active. Low-level expression of the recombinant bFSH is the factor that limits its usefulness as a superovulation treatment for cows. To increase the production of recombinant bFSH, human protein disulfide isomerase (hPDI) was expressed simultaneously in engineered Pichia strains. The secretion characteristics of bFSH with or without hPDI were examined. The co-expression of bFSH and hPDI is increased to 1.56 mg/l of heterodimer in the culture medium, which is 6-fold higher when compared with the control strain carrying the bFSH gene only. These results may be generally applicable to increase the expression of other glycoprotein hormones in yeast.     

A combined HNCA/HNCO experiment for 15N labeled proteins with 13C at natural abundance

A triple resonance NMR experiment is presented for the simultaneous recording of HNCA and HNCO data sets on ¹⁵N, natural abundance ¹³C samples. The experiment exploits the fact that transfers of magnetization from ¹⁵N to ¹³CO and from ¹⁵N to ¹³C (and back) proceed independently for samples that are not enriched in ¹³C. A factor of 2 in measuring time is gained by recording the two data sets simultaneously with no compromise in spectral quality. An application to a 0.5 mM ¹⁵N labeled sample of protein-L is presented with all expected correlations observed in spectra recorded with a cryogenic probe at 500 MHz.     

Cloning, sequence analysis and heterologous expression in Pichia pastoris of a gene encoding a thermostable cellobiose dehydrogenase from Myriococcum thermophilum

We cloned and expressed a gene encoding a thermostable cellobiose dehydrogenase (CDH) from the thermophilic ascomycete Myriococcum thermophilum. The 2904 bp long open reading frame contained six introns located either close to the 5′- or 3′-end of the ORF. The corresponding cDNA of 2487 bp was cloned into the expression vector pPICZαB to achieve inducible heterologous expression and secretion of the recombinant flavocytochrome in the methylotrophic yeast Pichia pastoris. Transformants were selected on media with normal and 10-fold increased zeocin concentration, and selected clones were tested for inducible extracellular production of the recombinant oxidoreductase. The maximally obtained volumetric activity was 0.25 U/ml in YPM (rich) medium and 2.15 U/ml in production stage (minimal) medium in a fed-batch fermentation. Recombinant CDH was purified in two consecutive chromatographic steps leading to a final specific activity of up to 7.4 U/mg protein at 40 °C. Kinetic properties of the recombinant CDH were characterized and the temperature optimum for the recombinant CDH was determined at 63 °C. Certain properties of the sequence of MtCDH are discussed in context with thermal and proteolytic stability.     

A method for efficient isotopic labeling of recombinant proteins

A rapid and efficient approach for preparing isotopically labeled recombinant proteins is presented. The method is demonstrated for ¹³C labeling of the C-terminal domain of angiopoietin-2, ¹⁵N labeling of ubiquitin and for ²H/¹³C/¹⁵N labeling of the Escherichia coli outer-membrane lipoprotein Lpp-56. The production method generates cell mass using unlabeled rich media followed by exchange into a small volume of labeled media at high cell density. Following a short period for growth recovery and unlabeled metabolite clearance, the cells are induced. The expression yields obtained provide a fourfold to eightfold reduction in isotope costs using simple shake flask growths.     

Cloning, expression, and purification of lipoprotein-associated phospholipase A2 in Pichia pastoris

Lipoprotein-associated phospholipase A₂ (Lp-PLA₂) has been shown to play a crucial role in atherosclerosis, and has been proposed as a promising target for drug discovery. Here, we cloned the Lp-PLA ₂ gene from differentiated THP-1 cells, and inserted a carboxy-terminal His₆-tagged version of the gene into the pPIC9 Pichia expression vector. The Lp-PLA₂ fusion protein was successfully expressed in Pichia pastoris expression system and could be rapidly purified to apparent homogeneity using a single-step purification method. The activity of our recombinant Lp-PLA₂ was strong when [³H] PAF was used as a substrate, and the Lp-PLA₂ inhibitor SB435495 exhibited an inhibitory curve against the recombinant Lp-PLA₂ (IC₅₀=15.93±1 μM). This novel recombinant Lp-PLA₂ could prove useful as a screening model for Lp-PLA₂ inhibitors, and may facilitate further investigation of this protein in atherosclerosis.