A new approach on the purification of recombinant human soluble catechol-O-methyltransferase from an Escherichia coli extract using hydrophobic interaction chromatography

Passarinha, Luís A.; Bonifácio, Maria João; Soares-da-Silva, Patrı́cio; Queiroz, João A.

doi:10.1016/j.chroma.2007.06.002

Cited by 20 publications

(35 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Commercial E. coli BL21-star (Invitrogene, Carlsbad, USA) was used as the recombinant strain for hSCOMT over expression under the control of the IPTG (Sigma, Missouri, USA) inducible promoter and employing carbanecillin disodium salt (Sigma, Missouri, USA) supplementation as a selection marker. The fermentation conditions were previously described by our group [18].…”

Section: Recombinant Hscomt Expressionmentioning

confidence: 99%

Analysis of hSCOMT adsorption in bioaffinity chromatography with immobilized amino acids: The influence of pH and ionic strength

Costa¹,

Bonifácio²,

Queiroz³

et al. 2011

Journal of Chromatography B

Self Cite

View full text Add to dashboard Cite

Section: Recombinant Hscomt Expressionmentioning

confidence: 99%

Analysis of hSCOMT adsorption in bioaffinity chromatography with immobilized amino acids: The influence of pH and ionic strength

Costa¹,

Bonifácio²,

Queiroz³

et al. 2011

Journal of Chromatography B

Self Cite

View full text Add to dashboard Cite

“…The Champion pET101 Directional TOPO expression kit (Invitrogen Corporation, Carlsbad, CA, U.S.A.) was used for the expression of human SCOMT in its native form on Escherichia coli BL21(DE3) strains, and the process was carried out according to the manufacturer's instructions as described elsewhere [25].…”

Section: Construction Of the Expression Vector And Bacterial Strainmentioning

confidence: 99%

“…In addition, the pH was close to 7.2 without adjustment before sterilization, and all the components were mixed and dissolved in deionized water and autoclaved for 15 min at 120 o C. After autoclaving, a specific volume of a standard glucose solution was added under aseptic environment conditions to the medium in order to obtain the desired substrate concentration. For all flasks experiments and methods to obtain a suitable hSCOMT soluble preparation, the specific details of inoculation, fermentation, and recuperation steps can be found elsewhere [25].…”

Section: Flask Experimentsmentioning

confidence: 99%

“…In the past decades, recombinant soluble catechol-Omethyltransferase (SCOMT) has been produced in E. coli [6,25], in insect cells using a baculovirus expression system [34], and in mammalian cell cultures using expression vectors based on Epstein-Barr virus (a herpesvirus; [34]) and Simian virus 40 (a polyomavirus; [37]). In spite of all of the afore-mentioned systems having produced functional forms of the enzyme, the methods enable to produce up to 1 g of target protein.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Application of a Fed-Batch Bioprocess for the Heterologous Production of hSCOMT in Escherichia coli

Passarinha

Bonifácio²,

Queiroz³

2009

J.Microbiol.Biotechnol

View full text Add to dashboard Cite

In this paper, a fed-batch cultivation process in recombinant Escherichia coli BL21(DE3) bacteria, for the production of human soluble catechol-O-methyltransferase (hSCOMT), is presented. For the first time, a straightforward model is applied in a recombinant hSCOMT expression system and distinguishes an initial cell growth phase from a protein production phase upon induction. Specifically, the kinetic model predicts biomass, substrate, and product concentrations in the culture over time and was identified from a series of fed-batch experiments designed by testing several feed profiles. The main advantage of this model is that its parameters can be identified more reliably from distinct fed-batch strategies, such as glycerol pulses and exponential followed by constant substrate additions. Interestingly, with the limited amount of data available, the proposed model accomplishes satisfactorily the experimental results obtained for the three state variables, and no exhaustive process knowledge is required. The comparison of the measurement data obtained in a validation experiment with the model predictions showed the great extrapolation capability of the model presented, which could provide new complementary information for the COMT production system.

show abstract

“…The 'active fractions' from HIC were further analyzed for purity and immunoreactivity, respectively by SDS-PAGE and western Blotting (Laemmli, 1970;Passarinha et al, 2008). The methylating efficiency of recombinant hSCOMT was evaluated by measuring the amount of metanephrine formed from epinephrine as substrate, by an electrochemical HPLC system (Passarinha et al, 2006).…”

Section: Methodsmentioning

confidence: 99%

Assessment of COMT isolation by HIC using a dual salt system and low temperature

Nunes

Bonifácio

Queiroz

et al. 2010

Biomedical Chromatography

View full text Add to dashboard Cite

Sodium citrate (SC) and low temperatures between 7 and 5 degrees C are effective in suppressing aggregation of proteins and may be beneficial to be included during a purification process. In this work, we analyzed the application of dual salt system, ammonium sulfate (AS) and SC on binding and elution conditions of recombinant hSCOMT on typical HIC sorbents. Specifically in butyl and octyl supports, the use of, respectively, 300 mM AS/200 mM SC and 25 mM AS/25 mM SC in the loading buffer resulted in complete binding of COMT. Elution was obtained by decreasing the ionic strength to 0 M of salt. For the delineate goal, it also favorably increased the support chain length while a consequent decrease in the dual ionic strength was observed for hSCOMT retention. In the presence of dual salt systems octyl media exhibited classic HIC behavior, good protein selectivity, an excellent purification factor and reduced denaturation effects of hSCOMT observed with higher salt concentrations. Also the inclusion of temperature control during the elution step appears to be advantageous for greater activity recovery without enzyme aggregation. In fact, these results could allow the prediction of most stabilizing conditions for this termolabile enzyme on the chromatographic stage, regarding salt types and therefore effectiveness to improve HIC selectivity and desirable purity on the target fractions.

show abstract

A new approach on the purification of recombinant human soluble catechol-O-methyltransferase from an Escherichia coli extract using hydrophobic interaction chromatography

Cited by 20 publications

References 26 publications

Analysis of hSCOMT adsorption in bioaffinity chromatography with immobilized amino acids: The influence of pH and ionic strength

Analysis of hSCOMT adsorption in bioaffinity chromatography with immobilized amino acids: The influence of pH and ionic strength

Application of a Fed-Batch Bioprocess for the Heterologous Production of hSCOMT in Escherichia coli

Assessment of COMT isolation by HIC using a dual salt system and low temperature

Contact Info

Product

Resources

About