Process simulation for recombinant protein production: Cost estimation and sensitivity analysis for heparinase I expressed inEscherichia coli

Ernst, Steffen; Garro, Oscar Alfredo; Winkler, Stefan; Venkataraman, Ganesh; Langer, Robert; Cooney, Charles L.; Sasisekharan, Ram

doi:10.1002/(sici)1097-0290(19970320)53:6<575::aid-bit5>3.0.co;2-j

Cited by 39 publications

(17 citation statements)

References 17 publications

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“…In particular, substantial resources are applied to the development of recombinant protein production with P. pastoris (Calik et al 2015), the most widely used system as expression system for heterologous protein production (Gasser et al 2013). Purification of recombinant proteins, including cell lysis is commonly recognized among the most costly parts of entire production processes (Ernst et al 1997). Additionally, the cells are disrupted frequently under harsh conditions that can affect the product quality.…”

Section: Discussionmentioning

confidence: 99%

Autolysis of Pichia pastoris induced by cold

et al. 2017

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The production of recombinant biopharmaceutical proteins is a multi-billion dollar market. Protein recovery represents a major part of the production costs. Pichia pastoris is one of the microbial systems most used for the production of heterologous proteins. The use of a cold-induced promoter to express lytic enzymes in the yeast after the growth stage could reduce protein recovery costs. This study shows that a cold-shock can be applied to induce lysis of the yeast cells. A strain of P. pastoris was constructed in which the endogenous eng gene encoding a putative endo-β-1,3-glucanase was overexpressed using the cold-shock induced promoter of the cctα gene from Saccharomyces cerevisiae. In the transgenic P. pastoris, the expression of eng increased 3.6-fold after chilling the cells from 30 to 4 °C (cold-shock stage) followed by incubation for 6 h (eng expression stage). The culture was heated to 30 °C for 6 h (ENG synthesis stage) and kept at 37 °C for 24 h (lysis stage). After this procedure the cell morphology changed, spheroplasts were obtained and cellular lysis was observed. Thus, a clone of P. pastoris was obtained, which undergoes autolysis after a cold-shock.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-017-0397-y) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Autolysis of Pichia pastoris induced by cold

et al. 2017

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“…Table 1 also shows the estimated cost for the production of 1 mg IFN-α2b using E. coli fermentation and transgenic tobacco plant systems. Production of IFN-α2b using E. coli is calculated based on two different working volumes of bioreactor (2.9 m 3 and 36.2 m 3 ) according to the method of Ernst et al [41]. Production of IFN-α2b using transgenic plants, however, is calculated based on Burley tobacco using the method of Foreman [42].…”

Section: Discussionmentioning

confidence: 99%

Comparison of expression systems for the production of human interferon-α2b

Memari¹,

Ramanan

Ariff³

2010

Open Life Sciences

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Abstract:The production of human interferon alpha2b (IFN-α2b) in two expression systems, tobacco (Nicotiana tabaccum) and Escherichia coli, was compared in various aspects such as safety, yield, quality of product and productivity. In the E. coli system, IFN-α2b was expressed under a pelB signal sequence and a T7lac promoter in a pET 26b(+) vector. The same gene was also cloned in expression plant vector (pCAMBIA1304) between cauliflower mosaic virus promoter (CaMV35S) and poly A termination region (Nos) and expressed in transgenic tobacco plants. The expression of protein in both systems was confirmed by western immunoblotting and the quantity of the protein was determined by immunoassay. The amount of periplasmic expression in E. coli was 60 µg/L of culture, while the amount of nuclear expression in the plant was 4.46 µg/kg of fresh leaves. The result of this study demonstrated that IFN-α2b was successfully expressed in periplasm of bacterial and plant systems. The limitations on the production of IFN-α2b by both systems are addressed and discussed to form the basis for the selection of the appropriate expression platform.© Versita Sp. z o.o.

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“…The ChnA and ChnB expression levels reached approximately 3.5 and 3.7%, in strains FIBX6 and FIBX7 grown in heparin-only medium, respectively. In E. coli, the recombinant HepI level reached 250 mg/liter when expressed as inclusion bodies or 150 mg/liter in a soluble form in a small-scale fermentation setting (5). In a 3-day FIBX5 fermentation, the productivity of HepI averaged 500 mg/liter but could reach a much higher level when the fermentation time was extended (B. Eggimann and H. Su, personal communication).…”

Section: Discussionmentioning

confidence: 99%

Expression System for High Levels of GAG Lyase Gene Expression and Study of the hepA Upstream Region in Flavobacterium heparinum

et al. 2002

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is described. hepA, along with its regulatory region, as well as hepB, hepC, cslA, and cslB, cloned downstream of the hepA regulatory region, was integrated in the chromosome to yield stable transconjugant strains. The level of heparinase I and II expression from the transconjugant strains was approximately fivefold higher, while heparinase III expression was 10-fold higher than in wild-type F. heparinum grown in heparin-only medium. The chondroitinase AC and B transconjugant strains, grown in heparin-only medium, yielded 20-and 13-fold increases, respectively, in chondroitinase AC and B expression, compared to wild-type F. heparinum grown in chondroitin sulfate A-only medium. The hepA upstream region was also studied using cslA as a reporter gene, and the transcriptional start site was determined to be 26 bp upstream of the start codon in the chondroitinase AC transconjugant strain. The transcriptional start sites were determined for hepA in both the wild-type F. heparinum and heparinase I transconjugant strains and were shown to be the same as in the chondroitinase AC transconjugant strain. The five GAG lyases were purified from these transconjugant strains and shown to be identical to their wild-type counterparts.

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Process simulation for recombinant protein production: Cost estimation and sensitivity analysis for heparinase I expressed inEscherichia coli

Cited by 39 publications

References 17 publications

Autolysis of Pichia pastoris induced by cold

Autolysis of Pichia pastoris induced by cold

Comparison of expression systems for the production of human interferon-α2b

Expression System for High Levels of GAG Lyase Gene Expression and Study of the hepA Upstream Region in Flavobacterium heparinum

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