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71.
Jan Heyland Jianan Fu Lars M. Blank Andreas Schmid 《Biotechnology and bioengineering》2010,107(2):357-368
Pichia pastoris has become one of the major microorganisms for the production of proteins in recent years. This development was mainly driven by the readily available genetic tools and the ease of high‐cell density cultivations using methanol (or methanol/glycerol mixtures) as inducer and carbon source. To overcome the observed limitations of methanol use such as high heat development, cell lysis, and explosion hazard, we here revisited the possibility to produce proteins with P. pastoris using glucose as sole carbon source. Using a recombinant P. pastoris strain in glucose limited fed‐batch cultivations, very high‐cell densities were reached (more than 200 gCDW L?1) resulting in a recombinant protein titer of about 6.5 g L?1. To investigate the impact of recombinant protein production and high‐cell density fermentation on the metabolism of P. pastoris, we used 13C‐tracer‐based metabolic flux analysis in batch and fed‐batch experiments. At a controlled growth rate of 0.12 h?1 in fed‐batch experiments an increased TCA cycle flux of 1.1 mmol g?1 h?1 compared to 0.7 mmol g?1 h?1 for the recombinant and reference strains, respectively, suggest a limited but significant flux rerouting of carbon and energy resources. This change in flux is most likely causal to protein synthesis. In summary, the results highlight the potential of glucose as carbon and energy source, enabling high biomass concentrations and protein titers. The insights into the operation of metabolism during recombinant protein production might guide strain design and fermentation development. Biotechnol. Bioeng. 2010;107: 357–368. © 2010 Wiley Periodicals, Inc. 相似文献
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74.
为实现人层粘连蛋白α4链LG4-5组件(Human Laminin Alpha4Chain LG4-5Module,hLNα4LG4-5)蛋白的分泌表达,采用DNA重组技术将hLNα4LG4-5cDNA片段插入分泌型酵母表达载体pPICZαA中,构建了相应的重组表达质粒pPICZαA-LG45并在GS115毕赤酵母菌株中表达,纯化蛋白后进行细胞学实验证明,hLNα4LG4-5有明显促进肿瘤细胞粘附和扩展的作用,为深入研究LG4-5组件结构与功能奠定了基础。 相似文献
75.
To obtain human tissue inhibitor of metalloproteinase-2 (TIMP-2)cDNA and the secretory expression of TIMP-2 gene in Pichia pastoris,we designed and synthesized a 618 base pairs artificial gene coding for the TIMP-2 with a computer-aided design method using a standard chemical synthesis technique,which was composed of frequently used codons in the highly expressed Pichia pastoris genes.Then the synthetic gene encoding TIMP-2 was checked by means of dideoxynucleotide sequencing.The verified gene of TIMP-2 was cloned to the Escherichia coli-yeast shuttle vector of pPIC9 to construct a recombinant plasmid pPIC9-T2.The plasmid was transformed into GS115 cells of the methylotrophic yeast,Pichia pastoris by electroporation,and we got the expression cell through phenotype selection and induction with methanol.Separation,purification,and bioactivity analysis of the expressed products were performed. 相似文献
76.
Dragosits M Frascotti G Bernard-Granger L Vázquez F Giuliani M Baumann K Rodríguez-Carmona E Tokkanen J Parrilli E Wiebe MG Kunert R Maurer M Gasser B Sauer M Branduardi P Pakula T Saloheimo M Penttilä M Ferrer P Luisa Tutino M Villaverde A Porro D Mattanovich D 《Biotechnology progress》2011,27(1):38-46
Microorganisms encounter diverse stress conditions in their native habitats but also during fermentation processes, which have an impact on industrial process performance. These environmental stresses and the physiological reactions they trigger, including changes in the protein folding/secretion machinery, are highly interrelated. Thus, the investigation of environmental factors, which influence protein expression and secretion is still of great importance. Among all the possible stresses, temperature appears particularly important for bioreactor cultivation of recombinant hosts, as reductions of growth temperature have been reported to increase recombinant protein production in various host organisms. Therefore, the impact of temperature on the secretion of proteins with therapeutic interest, exemplified by a model antibody Fab fragment, was analyzed in five different microbial protein production hosts growing under steady-state conditions in carbon-limited chemostat cultivations. Secretory expression of the heterodimeric antibody Fab fragment was successful in all five microbial host systems, namely Saccharomyces cerevisiae, Pichia pastoris, Trichoderma reesei, Escherichia coli and Pseudoalteromonas haloplanktis. In this comparative analysis we show that a reduction of cultivation temperature during growth at constant growth rate had a positive effect on Fab 3H6 production in three of four analyzed microorganisms, indicating common physiological responses, which favor recombinant protein production in prokaryotic as well as eukaryotic microbes. 相似文献
77.
Dale Fredericks Robert Clay Tracy Warner Anne O'Connor David M. de Kretser Milton T. W. Hearn 《Biotechnology progress》2010,26(2):372-383
We report a new procedure to express recombinant human activin A using the methanolic yeast, Pichia pastoris. Optimization of culture procedures has involved comprehensive examination of the effects of culture vessel shape, volume of broth in the induction and expression cultures, methanol concentration, culturing temperature, and pH of the expression cultures. After this optimization, as well as modification of the native cleavage sites, a laboratory scale procedure has been established which routinely produced 2–10 mg/L amounts of this vital growth factor in the highly efficient, eukaryotic yeast system. This system avoids the need to produce this protein and similar TGF‐β proteins in mammalian cell lines which, in addition to being costly, produce many native binding partners of these cystine knot proteins, a factor which can dramatically affect yields of the target protein. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 相似文献
78.
Extracellular human granulocyte-macrophage colony stimulating factor (hGM-CSF) expression was studied under the control of
the GAP promoter in recombinant Pichia pastoris in a series of continuous culture runs (dilution rates from 0.025 to 0.2 h−1). The inlet feed concentration was also varied and the steady state biomass concentration increased proportionally demonstrating
efficient substrate utilization and constancy of the biomass yield coefficient (Yx/s) for a given dilution rate. The specific product formation rate (qP) showed a strong correlation with dilution rates demonstrating growth associated product formation of hGM-CSF. The volumetric
product concentration achieved at the highest feed concentration (4×) and a dilution rate of 0.2 h−1 was 82 mg l−1 which was 5-fold higher compared to the continuous culture run with 1× feed concentration at the lowest dilution rate thus
translating to a 40 fold increase in the volumetric productivity. The specific product yield (YP/X) increased slightly from 2 to 2.5 mg g−1, with increasing dilution rates, while it remained fairly invariant, for all feed concentrations demonstrating negligible
product degradation or feed back inhibition. The robust nature of this expression system would make it easily amenable to
scale up for industrial production. 相似文献
79.
Lower induction temperature and polyoxyethylene sorbitan monolaurate (Tween-20) were successfully used to inhibit the aggregation
of recombinant human consensus interferon-α mutant (cIFN) during Pichia pastoris fermentation. When the induction temperature was decreased from 30 to 20°C, the cIFN secreted into the medium was in the form
of monomers instead of aggregates. The maximum specific activity at 20°C was 4.04 times as high as that at 30°C. There was
no obvious effect on the cell growth at 20°C, but the total protein level was decreased. Similar inhibition effect on cIFN
aggregation was observed when 0.2 g l−1 Tween-20 was added during induction. Furthermore, there was a synergistic effect found between induction temperature and
Tween-20 on the inhibition of cIFN aggregation. The maximum specific activity with Tween-20 at 20°C was 19.9-fold higher than
that without Tween-20 at 30°C. 相似文献
80.
A new approach to the production of the recombinant SOD protein by methylotrophic <Emphasis Type="Italic">Pichia pastoris</Emphasis> 总被引:3,自引:0,他引:3
Yu P 《Applied microbiology and biotechnology》2007,74(1):93-98
The gene for the copper, zinc–superoxide dismutase (SOD) from the yeast Saccharomyces cerevisiae was cloned, characterized, and overexpressed in the methylotrophic Pichia pastoris. The sod gene sequence obtained is 465 bp and encodes 154 amino acid residues. The sod gene sequence was cloned into the pPIC9K vector, yielding pAB22. The linearized pAB22 DNA, digested with restriction enzyme
SacI, was transformed into the genome of the GS115 strain of yeast P. pastoris. The overexpressed SOD protein was shown to have immunologically biological activity and to be enzymatically active. The
SOD protein was purified from the cultured yeast by ammonium sulfate precipitation and diethylaminoethyl–cellulose column
chromatography. This relatively simple purification method produced a single band on analysis by sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE), which indicated that the SOD protein obtained attained to higher purity and specific activity. 相似文献