Effect of Glycine and Triton X-100 on secretion and expression of ZZ–EGFP fusion protein

Liu

Appl Biochem Biotechnol

et al. 2011

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.

Section: Effect Of Glycine On Translocation Of Recombinant Cutinase Fmentioning

confidence: 94%

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

Liu

Appl Biochem Biotechnol

et al. 2011

Journal of Industrial Microbiology and Biotechnology

“…Glycine has been reported to improve the secretion of protein from E. coli through enhancement of cell membrane permeability [9,16,26]. To further improve the production of enzymatically active OPH, we therefore added different final concentrations of glycine to the culture medium at 24 h after culture initiation (the middle of the logarithmic phase, OD 600 = 13).…”

Section: Improvement Of Oph Expression By the Addition Of Glycinementioning

confidence: 99%

Expression and fermentation optimization of oxidized polyvinyl alcohol hydrolase in E. coli

Yang

Zhang

Liu

et al. 2012

Oxidized polyvinyl alcohol (PVA) hydrolase (OPH) is a key enzyme in the degradation of PVA, suggesting that OPH has a great potential for application in textile desizing processes. In this study, the OPH gene from Sphingopyxis sp. 113P3 was modified, by artificial synthesis, for overexpression in Escherichia coli. The OPH gene, lacking the sequence encoding the original signal peptide, was inserted into pET-20b (+) expression vector, which was then used to transform E. coli BL21 (DE3). OPH expression was detected in culture medium in which the transformed E. coli BL21 (DE3) was grown. Nutritional and environmental conditions were investigated for improved production of OPH protein by the recombinant strain. The highest OPH activity measured was 47.54 U/mL and was reached after 84 h under optimal fermentation conditions; this level is 2.64-fold higher that obtained under sub-optimal conditions. The productivity of recombinant OPH reached 565.95 U/L/h. The effect of glycine on the secretion of recombinant OPH was examined by adding glycine to the culture medium to a final concentration of 200 mM. This concentration of glycine reduced the fermentation time by 24 h and increased the productivity of recombinant OPH to 733.17 U/L/h. Our results suggest that the recombinant strain reported here has great potential for use in industrial applications.

“…In addition, some chemical additives play a useful role in increasing the solubility of recombinant proteins in E. coli [19]. Triton X-100 and Tween-80 are surfactants that can change the osmotic pressure in bacterial membranes [35]. Moreover, Triton X-100 supplementation in culture medium can hinder the formation of inclusion bodies in E. coli , and facilitate the secretion of recombinant proteins to the periplasmic space or culture medium [36].…”

Section: Discussionmentioning

confidence: 99%

Expression of a fungal manganese peroxidase in Escherichia coli: a comparison between the soluble and refolded enzymes

et al. 2016

BackgroundManganese peroxidase (MnP) from Irpex lacteus F17 has been shown to have a strong ability to degrade recalcitrant aromatic pollutants. In this study, a recombinant MnP from I. lacteus F17 was expressed in Escherichia coli Rosetta (DE3) in the form of inclusion bodies, which were refolded to achieve an active enzyme. Further, we optimized the in vitro refolding conditions to increase the recovery yield of the recombinant protein production. Additionally, we attempted to express recombinant MnP in soluble form in E. coli, and compared its activity with that of refolded MnP.ResultsRefolded MnP was obtained by optimizing the in vitro refolding conditions, and soluble MnP was produced in the presence of four additives, TritonX-100, Tween-80, ethanol, and glycerol, through incubation at 16 °C. Hemin and Ca2+ supplementation was crucial for the activity of the recombinant protein. Compared with refolded MnP, soluble MnP showed low catalytic efficiencies for Mn2+ and H2O2 substrates, but the two enzymes had an identical, broad range substrate specificity, and the ability to decolorize azo dyes. Furthermore, their enzymatic spectral characteristics were analysed by circular dichroism (CD), electronic absorption spectrum (UV-VIS), fluorescence and Raman spectra, indicating the differences in protein conformation between soluble and refolded MnP. Subsequently, size exclusion chromatography (SEC) and dynamic light scattering (DLS) analyses demonstrated that refolded MnP was a good monomer in solution, while soluble MnP predominantly existed in the oligomeric status.ConclusionsOur results showed that two forms of recombinant MnP could be expressed in E. coli by varying the culture conditions during protein expression.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0317-2) contains supplementary material, which is available to authorized users.