Very high-level production and export inEscherichia coli of a cellulose binding domain for use in a generic secretion-affinity fusion system

Hasenwinkle, D.; Jervis, Eric; Kops, Oliver; Liu, Chang; Lesnicki, Gary; Haynes, Charles A.; Kilburn, Douglas G.

doi:10.1002/(sici)1097-0290(19970920)55:6<854::aid-bit4>3.0.co;2-f

Cited by 26 publications

(13 citation statements)

References 46 publications

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“…The accumulation of recombinant protein in periplasm may cause an osmotic pressure build up to drive selective transport across the outer membrane [31]. Increase of outer membrane permeability is considered to be an effective method to facilitate protein translocation to culture medium on the premise of minimum inhibition of cell growth.…”

Section: Discussionmentioning

confidence: 99%

Study on Improvement of Extracellular Production of Recombinant Thermobifida fusca Cutinase by Escherichia coli

Chen

Liu

Chen

et al. 2011

Appl Biochem Biotechnol

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Escherichia coli is one of the most commonly used host strains for recombinant protein production. More and more research works on the production of recombinant protein indicate that extracellular production throughout a culture medium is more convenient and attractive compared to intracellular production. In present work, inducing temperature and isopropyl β-D: -1-thiogalactopyranoside (IPTG) concentration were investigated to decrease the formation of inclusion body and increase the amount of soluble recombinant cutinase initially. Enzyme activity in the culture medium reached to 118.9 U/ml at 64 h of culture, and no inclusion body was detected in cytoplasm under the inducement condition of 0.2 mM IPTG and 30°C. In addition, it was found that a large amount of cutinase had been accumulated in periplasm since 16-h cultivation under the same inducement condition. Therefore, glycine and surfactant sodium taurodeoxycholate (TDOC) were further used to promote the leakage of recombinant cutinase from periplasm. Supplied with 100 mM glycine and 1 mM TDOC, the amount of cutinase in periplasm decreased remarkably, and the activity in the culture medium reached to 146.2 and 149.2 U/ml after 54 h of culturing, respectively.

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Section: Discussionmentioning

confidence: 99%

Study on Improvement of Extracellular Production of Recombinant Thermobifida fusca Cutinase by Escherichia coli

Chen

Liu

Chen

et al. 2011

Appl Biochem Biotechnol

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“…The remainder was produced by fermentation. The gene fragment encoding CBM2a was subcloned into the pTugK07 vector and expressed in Escherichia coli strain JM101 according to the protocols of Hassenwinkle et al (18). The pTugK vector contains the leader sequence for xylanase 10A of C. fimi , which directs the recombinant protein to the periplasm of E. coli .…”

Section: Methodsmentioning

confidence: 99%

“…The pTugK vector contains the leader sequence for xylanase 10A of C. fimi , which directs the recombinant protein to the periplasm of E. coli . Hassenwinkle et al (18) demonstrated that significant leakage of CBM2a from the periplasm into the culture supernatant occurs at high levels of expression. CBM2a used in this work was therefore recovered from the culture supernatant by centrifuging the final culture at 4 °C and 10,000 rpm for 20 min and discarding the cell paste.…”

Section: Methodsmentioning

confidence: 99%

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Inexpensive and Generic Affinity Purification of Recombinant Proteins Using a Family 2a CBM Fusion Tag

et al. 2004

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The selective binding of the family 2a carbohydrate binding module (CBM2a) of xylanase 10A of the soil bacterium Cellulomonas fimi to a variety of cellulosic substrates is shown to provide a new, cost-effective affinity chromatography system for purification of recombinant protein. Genetic linkage of CBM2a to a target protein, in this case protein A from Staphylococcus aureus, results in a fusion protein that binds strongly to the particulate-cellulose resin Avicel PH101 and retains the biological activity of the fusion partner. Affinity purification of protein A-CBM2a from the supernatant of a recombinant E. coli JM101 culture results in a product purity of greater than 95% and a product concentration factor of 34 +/- 3. Measured column parameters are combined with one-dimensional equations governing continuity and intraparticle diffusion to predict product breakthrough curves with good accuracy over the range of realistic operating conditions. Peak spreading within the column is controlled by intraparticle diffusion for CBM2a and by a combination of film mass transfer and intraparticle diffusion for the larger protein A-CBM2a fusion protein.

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“…Consolidated bioprocessing (CBP) can be achieved by two alternative approaches: engineering cellulolytic organisms for high ethanol production, and engineering ethanologenic organisms to produce cellulase enzymes for biomass hydrolysis. Ethanologenic bacteria, such as Z. mobilis, E. coli and K. oxytoca, have been engineered for CBP by heterologous expression of cellulolytic enzymes [135][136][137][138]. The largest ethanol productivities were achieved with T. saccharolyticum by knocking out genes in organic acid synthesis pathways.…”

Section: Consolidated Bioprocessingmentioning

confidence: 99%

Engineering bacterial processes for cellulosic ethanol production

Kambam

Henson

2010

Biofuels

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Very high-level production and export inEscherichia coli of a cellulose binding domain for use in a generic secretion-affinity fusion system

Cited by 26 publications

References 46 publications

Study on Improvement of Extracellular Production of Recombinant Thermobifida fusca Cutinase by Escherichia coli

Study on Improvement of Extracellular Production of Recombinant Thermobifida fusca Cutinase by Escherichia coli

Inexpensive and Generic Affinity Purification of Recombinant Proteins Using a Family 2a CBM Fusion Tag

Engineering bacterial processes for cellulosic ethanol production

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