Expression level of heterologous proteins in Pichia pastoris is influenced by flask design

Villatte, Francois; As, Hussein; Tt, Bachmann; Rd, Schmid

doi:10.1007/s002530000479

Cited by 39 publications

(27 citation statements)

References 11 publications

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“…Production of large amounts of heterologous proteins in shake-flask culture is difficult, due to the limitations of volume, oxygen transfer, substrate addition and an inability to monitor these factors efficiently. Methods of monitoring methanol in shake flasks are available, such as organic solvent vapour detectors [40], and the oxygen transfer of these systems can be increased by the use of baffles [114]. On-line measurement of oxygen and carbon dioxide transfer rates in shake flasks has also been demonstrated using a respiration activity monitoring system (RAMOS) [100].…”

Section: Production Of Heterologous Proteins In P Pastorismentioning

confidence: 99%

Heterologous protein production using the Pichia pastoris expression system

Macauley-Patrick

Fazenda

McNeil

et al. 2005

Yeast

1,095

633

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The Pichia pastoris expression system is being used successfully for the production of various recombinant heterologous proteins. Recent developments with respect to the Pichia expression system have had an impact on not only the expression levels that can be achieved, but also the bioactivity of various heterologous proteins. We review here some of these recent developments, as well as strategies for reducing proteolytic degradation of the expressed recombinant protein at cultivation, cellular and protein levels. The problems associated with post-translational modifications performed on recombinant proteins by P. pastoris are discussed, including the effects on bioactivity and function of these proteins, and some engineering strategies for minimizing unwanted glycosylations. We pay particular attention to the importance of optimizing the physicochemical environment for efficient and maximal recombinant protein production in bioreactors and the role of process control in optimizing protein production is reviewed. Finally, future aspects of the use of the P. pastoris expression system are discussed with regard to the production of complex membrane proteins, such as G protein-coupled receptors, and the industrial and clinical importance of these proteins.

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Section: Production Of Heterologous Proteins In P Pastorismentioning

confidence: 99%

Heterologous protein production using the Pichia pastoris expression system

Macauley-Patrick

Fazenda

McNeil

et al. 2005

Yeast

1,095

633

View full text Add to dashboard Cite

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“…With P. pastoris systems, it has been shown that this increase in expression is strongly dependent on the extent of aeration and the shape of the baffles used. 58 Interestingly, it has been observed that dramatic differences in the expression levels of acetylcholinesterase occur with flasks with different shaped baffles, but these expression levels do not correlate with oxygenation levels in these flasks, suggesting the other factors besides oxygenation influence expression levels in shakeflask cultures. 58 As anticipated, the addition of half concentrations of methanol for every 12 h gave enhanced levels of expression of activin A.…”

Section: Larger Scale Expression Studiesmentioning

confidence: 98%

Optimization of the expression of recombinant human activin A in the yeast Pichia pastoris

Fredericks

Clay

Warner

et al. 2010

Biotechnology Progress

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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-beta 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.

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“…Although it has been reported that P. pastoris represents an appropriate host for the expression of recombinant proteins, the low level of expression in this host could be attributed to several factors. These potential impediments include, the lack of consistency of codon usage of the bovine and pichia (8), copy number of the gene (9), the efficiency and strength of promoters (10), efficiency of translation signals (11) and signal peptides (12), processing and folding in the endoplasmic reticulum and Golgi apparatus (13), environmental factors of expression (14), extracellular secretion (15), and protein turnover by proteolysis (16,17). As the recombinant, colony number 6 is high geneticin resistant (which grown on 2 mg/mL of this Antibiotic), the copy number of the gene was not restrictive for recombinant protein production.…”

Section: Discussionmentioning

confidence: 99%