Recombinant protein production data after expression in the bacterium Escherichia coli

Cantu-Bustos, J. Enrique; Villar, Kevin D. Cano del; Vargas‐Cortez, Teresa; Morones‐Ramírez, José Rubén; Balderas-Renterı́a, Isaı́as; Zárate, Xristo

doi:10.1016/j.dib.2016.02.074

Cited by 5 publications

(4 citation statements)

References 13 publications

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“…When the cultivation period ended, cells were harvested by centrifugation at rpm for 10 min (Benchtop centrifuge, Thermo Scientific, MA, USA). The bacterial pellet was resuspended in lysis buffer (5 mL/g of cell pellet) containing 20 m Tris HCl pH 8, NaCl 250 mM, glycerol 5%, DNase, 0,3% v/v of triton X-100, lysozyme 60 µg/mL, PMSF 0.5 mM and incubated at 37 °C for 40 min [69,70]. The suspension was centrifuged at 13000 rpm for min at 5°C to obtain the soluble and insoluble fraction (Inclusion bodies).…”

Section: Effect Of Osmolytes Glycerol Sorbitol and Glycine On Recombmentioning

confidence: 99%

Optimization of Fermentation Conditions for Recombinant Lipase Expression At Small Scale Using Response Surface Methodology for Preliminary Production In Two Bioreactor Platforms.

López

Acosta-González

Manrique

et al. 2021

Preprint

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Background: Pseudomonas lipases are widely used in industrial applications due to its unique biochemical properties, but one of the biggest limitations are the low yields obtained in native strains therefore, organisms as E. coli are used for the recombinant lipase overexpression. However, the recombinant lipase is accumulated as inclusion bodies and it affects biological activity, making that researchers evaluate different fermentation conditions to improve the activity of recombinant enzymes. In this study, a statistical experimental design was implemented to evaluate the effects of temperature, agitation rate and osmolyte concentration on the recombinant lipase activity produced in E. coli BL21 (DE3). Once the significant variables were identified, an optimization by a Response Surface Methodology was applied to maximize the lipase production. Results: The Box-Behnken designs revealed different optimal fermentation conditions for each osmolyte experiment. The glycerol showed the highest specific lipase activity compared to the other osmolytes and 0.1 M of osmolyte glycerol,5°C and 110 rpm showed the highest significant increase on the specific lipase activity and the data fitted the model very well. The validation showed that 452.01 U/mg of specific lipase activity was obtained which was significantly higher compared to the group where no glycerol was added (271.38 U/mg). The relative recombinant lipase expression was 2.7-fold lower at 5°C compared to 25 °C, but at 5°C the lipase activity was significantly higher. In addition, when the 3 L shaken Erlenmeyer Bioreactor was used to produce the recombinant lipase based on the power input parameter, the specific lipase activity was not significantly different from that found in Schott (408,4 U/mg and 452 U/mg, respectively), which means that this Bioreactor platform should be used for future scale-up processes.Conclusion: Low temperatures, low agitation rates and 0.1 M of glycerol in the autoinduction media enhanced the activity of the recombinant lipase produced in E. coli BL21(DE3). The optimized conditions and the 3 L shaken Erlenmeyer Bioreactor can be used to produce the recombinant enzyme in a higher volume based on the power input parameter. Further studies using this strategy may lead to the identification of optimal culture conditions for a given recombinant enzyme facilitating the large-scale bioprocess implementation.

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Section: Effect Of Osmolytes Glycerol Sorbitol and Glycine On Recombmentioning

confidence: 99%

Optimization of Fermentation Conditions for Recombinant Lipase Expression At Small Scale Using Response Surface Methodology for Preliminary Production In Two Bioreactor Platforms.

López

Acosta-González

Manrique

et al. 2021

Preprint

View full text Add to dashboard Cite

show abstract

“…The purity of the rhASP was measured by the reverse phase-high pressure liquid chromatography (RP-HPLC) 20 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 21 The secondary structural confirmation of purified rhASP was evaluated by the circular dichroism (CD) spectroscopy (AVIV 420SF spectropolarimeter, NJ) using a 2 mm path length at a scan rate of 20 nm/min. Bradford method was used to estimate the concentration of the rhASP with Bovine serum albumin (BSA) as a protein standard.…”

Section: Qualitative and Quantitative Proceduresmentioning

confidence: 99%

Efficient and easily scalable protein folding strong anion exchange chromatography for renaturation and simultaneous purification of recombinant human asparaginase from E. coli

Kante

Vemula

Mallu

et al. 2018

Biotechnology Progress

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Recombinant proteins are revolutionizing present day therapeutics. They are generally expressed as insoluble inclusion bodies in the E. coli and mis-folding, loss of protein, and high cost of down streaming are the hurdles in their recovery. For the first time, we are reporting the refolding with simultaneous purification of rhASP in E. coli using a single step utilizing protein folding-strong anion exchange chromatography (PF-SAX). The purification method is also standardized for optimal concentration of solution additives, pH, and mobile phase composition. The results showed purification of rhASP with anion exchange chromatography was effective. Phosphate buffer and slightly alkaline pH produced significant recovery yields and purity profiles. The effect of solution additives such as arginine, glycerol, TMAO, sorbitol, dextran, glutamate, and fructose on rhASP renaturation is also investigated. Significant results were achieved using arginine-TMAO combination in terms of purity, recovery yield and specific activity of 99%, 78%, and 210 IU/mg, respectively. The work concludes that PF-SAX refolding method is superior to other conventional methods and it can be applied to large scale purification of rhASP produced in E. coli. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1036-1044, 2018.

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“…Recombinant therapeutic proteins are converted to their native form by removal of N-terminal methionine residue (Arif et al, 2015;Calcagno and Klein, 2016). Because of its easy manipulation, cultivation, high yield, and better economy, the E. coli strains include the most extensively used protein expression systems (Elleuche et al, 2015;Cantu-Bustos et al, 2016;Chen et al, 2016;Jia and Jeon, 2016). Once produced in E. coli, activity of aminopeptidase is one of many decisive factors for a recombinant protein to exhibit a replica of its native form (Tripathi, 2016).…”

Section: Introductionmentioning

confidence: 99%

Biochemical and in silico evaluation of recombinant E. coli aminopeptidase and in vitro processed human interferon α-2b

2018

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Escherichia coli is an extensively used host for the production of recombinant proteins, making its N-terminal methionine aminopeptidase (MAP) an attractive candidate for studies on posttranslational protein processing. The present study describes the recombinant production and properties of MAP from the DH5α strain of E. coli. The soluble and active enzyme was produced in E. coli BL21 (DE3) RIL -codon plus cells under a T7 promoter system and purified by anion-exchange chromatography. It exhibited a molecular weight of 29,200.94 Da by MALDI-TOF analysis. The purified enzyme showed specific activity of 1.64 U/mg with methionylp-nitroanilide and 1.51 U/mg with synthetic tetrapeptide substrate 'MGMM' in a discontinuous HPLC-based assay. In vitro studies showed the processing of up to 36% of Met-INFα-2b in 40 min. In silico studies revealed that the ES-complex formation between the enzyme and interferon has a ΔG -683.07 kJ/mol. Molecular docking results showed that the processed INFα-2b has greater binding affinity with IFNAR2 receptor as indicated by ΔG -784.53 kJ/mol, significantly lower than that of methionine containing INFα-2b (ΔG -717.63 kJ/mol). These findings emphasize the functional superiority or better efficacy of N-terminal methionine processed recombinant interferon.

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Recombinant protein production data after expression in the bacterium Escherichia coli

Cited by 5 publications

References 13 publications

Optimization of Fermentation Conditions for Recombinant Lipase Expression At Small Scale Using Response Surface Methodology for Preliminary Production In Two Bioreactor Platforms.

Optimization of Fermentation Conditions for Recombinant Lipase Expression At Small Scale Using Response Surface Methodology for Preliminary Production In Two Bioreactor Platforms.

Efficient and easily scalable protein folding strong anion exchange chromatography for renaturation and simultaneous purification of recombinant human asparaginase from E. coli

Biochemical and in silico evaluation of recombinant E. coli aminopeptidase and in vitro processed human interferon α-2b

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