Recombinant protein production in bacterial hosts

Overton, Tim W.

doi:10.1016/j.drudis.2013.11.008

Cited by 180 publications

(152 citation statements)

References 64 publications

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“…Currently expression of recombinant proteins in E. coli accounts for 30% of production of marketed pharmaceuticals [40], with the inability to carry out post-translational modifications causing it to lose its leading role to Chinese Hamster Ovary (CHO) cultures [41]. In contrast to bacterial cultures CHO have high nutritional requirements, grow slowly, are very fragile and instable; establishment and optimization of production strains for each protein of interest takes a long time correlating with high production costs [41].…”

Section: Discussionmentioning

confidence: 99%

Efficient soluble expression of disulfide bonded proteins in the cytoplasm of Escherichia coli in fed-batch fermentations on chemically defined minimal media

et al. 2017

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BackgroundThe production of recombinant proteins containing disulfide bonds in Escherichia coli is challenging. In most cases the protein of interest needs to be either targeted to the oxidizing periplasm or expressed in the cytoplasm in the form of inclusion bodies, then solubilized and re-folded in vitro. Both of these approaches have limitations. Previously we showed that soluble expression of disulfide bonded proteins in the cytoplasm of E. coli is possible at shake flask scale with a system, known as CyDisCo, which is based on co-expression of a protein of interest along with a sulfhydryl oxidase and a disulfide bond isomerase. With CyDisCo it is possible to produce disulfide bonded proteins in the presence of intact reducing pathways in the cytoplasm.ResultsHere we scaled up production of four disulfide bonded proteins to stirred tank bioreactors and achieved high cell densities and protein yields in glucose fed-batch fermentations, using an E. coli strain (BW25113) with the cytoplasmic reducing pathways intact. Even without process optimization production of purified human single chain IgA1 antibody fragment reached 139 mg/L and hen avidin 71 mg/L, while purified yields of human growth hormone 1 and interleukin 6 were around 1 g/L. Preliminary results show that human growth hormone 1 was also efficiently produced in fermentations of W3110 strain and when glucose was replaced with glycerol as the carbon source.ConclusionsOur results show for the first time that efficient production of high yields of soluble disulfide bonded proteins in the cytoplasm of E. coli with the reducing pathways intact is feasible to scale-up to bioreactor cultivations on chemically defined minimal media.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0721-x) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Efficient soluble expression of disulfide bonded proteins in the cytoplasm of Escherichia coli in fed-batch fermentations on chemically defined minimal media

et al. 2017

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“…Since then, the improvement of new heterologous protein production systems via E. coli enabled the commercial approval of several other products, including hormones (calcitonin, parathyroid hormone, human growth hormone, glucagon, and somatropin), interferons, and interleukins (Ferrer-Miralles et al, 2009). For example, approximately 30% of commercially available recombinant proteins are currently produced in prokaryotic systems (Overton, 2014). This production method is due to the unusual physiology of the cells as well as the ease of genetically manipulating them, but the understanding is that it is possible, by adding heterologous reactions, to synthesize 1,777 non-native products from E. coli , of which 279 have commercial applications.…”

Section: Medical Technological Microbiologymentioning

confidence: 99%

Technological Microbiology: Development and Applications

Vitorino

Bessa

2017

Front. Microbiol.

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Over thousands of years, modernization could be predicted for the use of microorganisms in the production of foods and beverages. However, the current accelerated pace of new food production is due to the rapid incorporation of biotechnological techniques that allow the rapid identification of new molecules and microorganisms or even the genetic improvement of known species. At no other time in history have microorganisms been so present in areas such as agriculture and medicine, except as recognized villains. Currently, however, beneficial microorganisms such as plant growth promoters and phytopathogen controllers are required by various agricultural crops, and many species are being used as biofactories of important pharmacological molecules. The use of biofactories does not end there: microorganisms have been explored for the synthesis of diverse chemicals, fuel molecules, and industrial polymers, and strains environmentally important due to their biodecomposing or biosorption capacity have gained interest in research laboratories and in industrial activities. We call this new microbiology Technological Microbiology, and we believe that complex techniques, such as heterologous expression and metabolic engineering, can be increasingly incorporated into this applied science, allowing the generation of new and improved products and services.

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“…Sequentially, in the fermentation process, how to better balance the cell growth and expression becomes another key problem . Much research suggested that the fermentation efficiency was greatly influenced by the induction conditions, cell growth and substrate inhibition (Donovan et al 1996;Pinsach et al 2008a;Overton 2014). It was reported that the induction conditions were closely related with cell growth and metabolic response (Gombert and Kilikian 1998;Chen et al 2013).…”

Section: Introductionmentioning

confidence: 98%

Novel integration strategy coupling codon and fermentation optimization for efficiently enhancing sarcosine oxidase (SOX) production in recombinant Escherichia coli

Tong

Yang

Xin

et al. 2015

World J Microbiol Biotechnol

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Sarcosine oxidase (SOX) was an important diagnostic enzyme in the renal function examination. An integrated strategy coupling codon and fermentation optimization was firstly proposed for improving SOX production from recombinant E. coli in 3-L fermentor. The expression suppression (gene phase) and poor balance between SOX expression and cell growth (fermentation phase) in the traditional SOX production were respectively improved by the multiple strategies. Based on the codon bias, the expression suppression was weakened via codon optimization and SOX activity reached 1,521 U/L. The induction toxicity was reduced with the optimal induction condition and SOX production increased to 4,015 U/L. Based on the kinetic analysis of μ x and μ p , a better balance between cell growth and expression was achieved by the two-stage pH-stat control strategy. The SOX activity was further improved to 8,490 U/L and fermentation cycle was also significantly shortened from 44 to 32 h. The substrate inhibition was weakened with a constant feeding fed-batch. With the assistance of integrated strategy, the activity and productivity reached 12,466 U/L and 389.6 U/(L h), respectively, or 3.1-fold and 4.3-fold of the uncontrolled fermentation. The strategy would be also useful in the industrial application of other similar enzymes.

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Recombinant protein production in bacterial hosts

Cited by 180 publications

References 64 publications

Efficient soluble expression of disulfide bonded proteins in the cytoplasm of Escherichia coli in fed-batch fermentations on chemically defined minimal media

Efficient soluble expression of disulfide bonded proteins in the cytoplasm of Escherichia coli in fed-batch fermentations on chemically defined minimal media

Technological Microbiology: Development and Applications

Novel integration strategy coupling codon and fermentation optimization for efficiently enhancing sarcosine oxidase (SOX) production in recombinant Escherichia coli

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