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2.
The methylotrophic yeast Pichia pastoris is known as an efficient host for the production of heterologous proteins. While N-linked protein glycosylation is well characterized in P. pastoris there is less knowledge of the patterns of O-glycosylation. O-glycans produced by P. pastoris consist of short linear mannose chains, which in the case of recombinant biopharmaceuticals can trigger an immune response in humans. This study aims to reveal the influence of different cultivation strategies on O-mannosylation profiles in P. pastoris. Sixteen different model proteins, produced by different P. pastoris strains, are analyzed for their O-glycosylation profile. Based on the obtained data, human serum albumin (HSA) is chosen to be produced in fast and slow growth fed batch fermentations by using common promoters, P GAP and P AOX1. After purification and protein digestion, glycopeptides are analyzed by LC/ESI-MS. In the samples expressed with P GAP it is found that the degree of glycosylation is slightly higher when a slow growth rate is used, regardless of the efficiency of the producing strain. The highest glycosylation intensity is observed in HSA produced with P AOX1. The results indicate that the O-glycosylation level is markedly higher when the protein is produced in a methanol-based expression system. 相似文献
4.
The gene encoding translation elongation factor 1-α from the yeast Pichia pastoris was cloned. The gene revealed an open reading frame of 1,380 bp with the potential to encode a polypeptide of 459 amino acids
with a calculated mass of 50.1 kDa. The potential of the promoter ( P
TEF1
) in P. pastoris was investigated with comparison to the glyceraldehyde-3-phosphate dehydrogenase promoter ( P
GAP
) by using a bacterial lipase gene as a reporter gene. P
TEF1
demonstrated a tighter growth-associated expression mode, improved functioning in the presence of high glucose concentrations,
and promoter activities that yielded recombinant protein at levels similar to or in one case greater than P
GAP
. The sequence of the gene was deposited in GenBank under accession no. EF014948. 相似文献
5.
The 3′-deleted amylopullulanase gene from the extreme thermophile Geobacillus thermoleovorans (Gt-apuΔC) was expressed extracellularly in Pichia pastoris under both methanol-inducible AOX1 and constitutive GAP promoters. The expression of the gene (Gt-apuΔC) was higher under GAP promoter (36.2 U ml−1, α-amylase; 33.5 U ml−1, pullulanase) than that under AOX1 promoter (32.5 and 28.6 U ml−1). The heavily glycosylated Gt-apuΔC from the recombinant P. pastoris displays higher substrate specificity, thermal stability and starch saccharification efficiency than that expressed in Escherichia coli. The enzyme hydrolyses maltotriose and maltotetraose unlike that expressed in E. coli. The enzyme action on wheat bran liberates maltose and glucose without detectable amount(s) of maltooligosaccharides. The sugars released from wheat bran (glucose and maltose) could be fractionated by ultrafiltration, as confirmed by TLC and HPLC analysis. This is the first report on the production of recombinant amylopullulanase extracellularly in P. pastoris.
相似文献
6.
BackgroundThe methanol-regulated AOX1 promoter (PAOX1) is the most widely used promoter in the production of recombinant proteins in the methylotrophic yeast Pichia pastoris. However, as the tight regulation and methanol dependence of PAOX1 restricts its application, it is necessary to develop a flexible induction system to avoid the problems of methanol without losing the advantages of PAOX1. The availability of synthetic biology tools enables researchers to reprogram the cellular behaviour of P. pastoris to achieve this goal.ResultsThe characteristics of PAOX1 are highly related to the expression profile of methanol expression regulator 1 (Mxr1). In this study, we applied a biologically inspired strategy to reprogram regulatory networks in P. pastoris. A reprogrammed P. pastoris was constructed by inserting a synthetic positive feedback circuit of Mxr1 driven by a weak AOX2 promoter (PAOX2). This novel approach enhanced PAOX1 efficiency by providing extra Mxr1 and generated switchable Mxr1 expression to allow PAOX1 to be induced under glycerol starvation or carbon-free conditions. Additionally, the inhibitory effect of glycerol on PAOX1 was retained because the synthetic circuit was not activated in response to glycerol. Using green fluorescent protein as a demonstration, this reprogrammed P. pastoris strain displayed stronger fluorescence intensity than non-reprogrammed cells under both methanol induction and glycerol starvation. Moreover, with single-chain variable fragment (scFv) as the model protein, increases in extracellular scFv productivity of 98 and 269% were observed in Mxr1-reprogrammed cells under methanol induction and glycerol starvation, respectively, compared to productivity in non-reprogrammed cells under methanol induction.ConclusionsWe successfully demonstrate that the synthetic positive feedback circuit of Mxr1 enhances recombinant protein production efficiency in P. pastoris and create a methanol-free induction system to eliminate the potential risks of methanol. 相似文献
7.
Background
Trigonopsis variabilis
D -amino acid oxidase ( Tv DAO) is a well characterized enzyme used for cephalosporin C conversion on industrial scale. However, the demands on the
enzyme with respect to activity, operational stability and costs also vary with the field of application. Processes that use
the soluble enzyme suffer from fast inactivation of Tv DAO while immobilized oxidase preparations raise issues related to expensive carriers and catalyst efficiency. Therefore,
oxidase preparations that are more robust and active than those currently available would enable a much broader range of economically
viable applications of this enzyme in fine chemical syntheses. A multi-step engineering approach was chosen here to develop
a robust and highly active Pichia pastoris Tv DAO whole-cell biocatalyst. 相似文献
9.
The promoter region of copper-inducible laccase gene, LCC1, from Pycnoporus coccineus was explored in the heterologous expression of foreign protein in Pichia pastoris. The promoter region (P PPLCC1) was isolated and used to replace the methanol-inducible AOXI promoter (P AOX1) of pPICHOLI-2, an episomal expression vector for P. pastoris, to generate a new copper-inducible expression vector. The promoter activity of P PPLCC1 was compared with those of P AOX1 and P CUP1, a copper-inducible promoter of a commercial vector pPICHOLI-C, using a laccase gene as a reporter gene in P. pastoris GS115. Reporter laccase activity of the culture broth reached 182 and 43 units/L for P PPLCC1 and P CUP1, respectively, after induction with 0.2 mM CuSO 4 at OD 600 = 1 and culture for 120 h at 15°C in complex medium containing 1% glucose. For P AOX1 activity, yeast cells harboring P AOX1-laccase plasmid were cultured for 120 h at 15°C in complex medium with intermittent feeding with 1% methanol every 12 h to avoid methanol toxicity. Laccase activity of culture broth was 124 units/L. Conclusively, P PPLCC1 is a new copper-inducible promoter that shows superior performance in terms of efficiency of laccase production compared to commercial vectors. P PPLCC1 is additionally superior to P AOX1 since it does not require laborious feeding with a carbon source. 相似文献
12.
A high-density cell culture method to produce human angiostatin has been successfully established by constitutive expression
of the protein in Pichia pastoris. The fermentation was carried out in a 20 l bioreactor with a 10 l working volume, using a high-density cell culture method
by continuously feeding with 50% glycerol−0.8% PTM4 to the growing culture for 60 h at 30°C. Dissolved oxygen level was maintained
at 25–30% and pH was controlled at 5 by the addition of 7 M NH 4OH. Angiostatin was constitutively expressed during the fermentation by linking its expression to the P. pastoris constitutive GAP promoter (pGAP). But after 36 h of fermentation, the peak biomass growth was 305 as measured by absorption
of 600 nm, while the peak angiostatin expression was 176 mg/l. Similar to the product expressed from inducible system [ 24], angiostatin produced from constitutive system also inhibited the angiogenesis on the CAM and suppressed the growth of B16
melanoma in C57BL/6J mouse. The above results suggest that GAP promoter is more efficient than AOX1 promoter for the expression
of angiostatin in P. pastoris by shake flask culture or high-density cell fermentation and is likely to be an alternative to AOX1 promoter in large-scale
expression of angiostatin and other heterologous proteins.
Supported by the Natural Science Foundation of China (39670013) and “225” Science and Technology Program of Guangzhou Municipal
Government of China (99-Z-004-001). 相似文献
13.
Hydrophobins are small surface‐active proteins that have considerable potential for use in applications ranging from medical and technical coatings, separation technologies, biosensors, and personal care. Their wider use would be facilitated by the availability of recombinant tailor‐made hydrophobins. We successfully expressed the class II hydrophobin HFB1 from Trichoderma reesei in Pichia pastoris under the control of the constitutive GAP (glyceraldehyde 3‐phosphate dehydrogenase) promoter. Avoiding the use of the AOX1 (alcohol oxidase 1) promoter prevents the costs and risks associated with the storage and delivery of methanol used as an inducer. Efficient secretion of hydrophobin was achieved using either the alpha‐factor prepro‐peptide or the native secretion signal of HFB1. The secreted hydrophobins have been isolated with a purity of up to 70% using in situ foam separation during the cultivation process. Coating experiments and surface pressure measurements demonstrated the activity of the hydrophobins. An immunodot assay showed the accessibility of carboxyterminally fused tags of the hydrophobin, which is necessary for potential applications using functionalized hydrophobins. The presented data show that Pichia pastoris is a suitable system for production of constitutively expressed and secreted active hydrophobin, allowing for in situ pre‐purification using foam separation. 相似文献
16.
A xylose reductase gene ( xyl1) of Candida guilliermondii ATCC 20118 was cloned and characterized. The open reading frame of xyl1 contained 954 nucleotides encoding a protein of 317 amino acids with a predicted molecular mass of 36 kDa. The derived amino
acid sequence of C. guilliermondii xylose reductase was 70.4% homologous to that of Pichia stipitis. The gene was placed under the control of an alcohol oxidase promoter ( AOX1) and integrated into the genome of a methylotrophic yeast, Pichia pastoris. Methanol induced the expression of the 36-kDa xylose reductase in both intracellular and secreted expression systems. The
expressed enzyme preferentially utilized NADPH as a cofactor and was functional both in vitro and in vivo. The different cofactor
specificity between P. pastoris and C. guilliermondii xylose reductases might be due to the difference in the numbers of histidine residues and their locations between the two
proteins. The recombinant was able to ferment xylose, and the maximum xylitol accumulation (7.8 g/l) was observed when the
organism was grown under aerobic conditions.
Received: 26 August 1997 / Received revision: 6 November 1997 / Accepted: 21 November 1997 相似文献
17.
The genes encoding the two enzymes choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH) of glycine betaine
synthesis in Suaeda salsa were cloned and fused with the 2A region of foot-and-mouth disease virus in a single open reading frame. The fused genes were placed under the control of the
alcohol oxidase ( AOX1) promoter in pPIC3B and transformed into P. pastoris GS115. The expression of the fused genes in P. pastoris and the ability of recombinant yeasts to tolerate environmental stresses were studied. The results showed that induced with
0.5% methanol for 96 h, the maximal activities of CMO and BADH in the tested recombinant yeasts were 45- and 44-fold higher
than those in the control yeast transformed empty vector only, respectively; the content of glycine betaine in the recombinant
yeasts was 28- to 35-fold higher than that in the control. The fused genes linked by 2A region of foot-and-mouth disease virus were expressed in P. pastoris successfully and the polyprotein was ‘cleaved’ to each functional protein. The yeasts transformed the fused genes, which
were more resistant to salt, methanol, and high temperature stresses than the control as result of glycine betaine synthesis
genes introduced. 相似文献
18.
The xylanase gene xyn II from Aspergillus usamii E001 was placed under the control of an alcohol oxidase promoter ( AOX1) in the plasmid pPIC9K and integrated into the genome of a methylotrophic yeast, P. pastoris GS115, by electroporation. His + transformants were screened for on the basis of their resistance to G418 and activity assay. A transformant, P. pastoris GSC12, which showed resistance to over 6 mg G418/ml and highest xylanase activity was selected. Recombinant xylanase was
secreted by P. pastoris GSC12 24 h after methanol induction of shake-flask cultures, and reached a final yield of 3139. About 68 U/mg 120 h after
the induction. The molecular mass of this xylanase was estimated to be 21 kDa by SDS-PAGE. The optimum pH and temperature
were 4.2 and 50 °C, respectively. Xylanase was stable below 50 °C and within pH 3.0–7.0. Its activity was increased by EDTA
and Co 2+ ion and strongly inhibited by Mn 2+, Li + and Ag + ions. The K
m and V
max values with birchwood xylan as the substrate were found to be 5.56 mg/ml and 216 μmol/mg/min, respectively. This is the first
report on expression and characterization of xylanase from A. usamii in P. pastoris. The hydrolysis products consisted of xylooligosaccharides together with a small amount of xylose. This property made the
enzyme attractive for industrial purposes, as relatively pure xylooligosaccharides could be obtained. 相似文献
19.
Angiotensinogen (AGT), a glycosylated plasma noninhibitory serpin, serves as a precursor for angiotensin peptides which regulate blood pressure and electrolyte balance. AGT is specifically cleaved by renin to produce angiotensin-I, the first product of the angiotensin-processing cascade. Ovine angiotensinogen (oAGT) is considered an effective substrate for human renin and consequently finds application in clinical renin assays. In this study, oAGT was cloned into the genome of Pichia pastoris and expressed under the control of alcohol oxidase (AOX1) promoter for high-level production. Compared to the shake flask study, the high cell density cultivation in bioreactor resulted in multifold increase in oAGT titer (420 ± 9.26 mg/L), which is its highest reported titer to date. We purified recombinant oAGT to homogeneity using two chromatography steps. The characterization studies revealed oAGT underwent a two-state transition during thermal denaturation process as assessed by differential scanning fluorimetry, and the melting temperature ( Tm) of the purified oAGT from P. pastoris was 48.3°C. Renin reactivity with recombinant oAGT from P. pastoris (0.51 nM angiotensin-I/min) was slightly lower than the renin reactivity for recombinant oAGT from Escherichia coli (0.67 nM angiotensin-I/min), possibly because of its mannosylated N-glycan content. Enhanced production of functionally active recombinant oAGT using P. pastoris expression system reported in this study envisage the effective utilization of oAGT in clinical studies related to renin in near future. 相似文献
20.
Pichia pastoris is an efficient host for the expression and secretion of heterologous proteins and the most important feature of P. pastoris is the existence of a strong and tightly regulated promoter from the alcohol oxidase I (AOX1) gene. The AOX1 promoter (pAOX1)
has been used to express foreign genes and to produce a variety of recombinant proteins in P. pastoris. However, some efforts have been made to develop new alternative promoters to pAOX1 to avoid the use of methanol. The glyceraldehyde-3-phosphate
dehydrogenase promoter (pGAP) has been used for constitutive expression of many heterologous proteins. The pGAP-based expression
system is more suitable for large-scale production because the hazard and cost associated with the storage and delivery of
large volume of methanol are eliminated. Some important developments and features of this expression system will be summarized
in this review.
Supported by the National High-tech R&D Program (863 program) (No.2007AA021307). 相似文献
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