DOI: 10.1002/bit.20806. Affinity‐tagged green fluorescent protein (GFP) extraction from a clarified <i>E. coli</i> cell lysate using a two‐phase aqueous micellar system

Affinity‐tagged green fluorescent protein (GFP) extraction from a clarified E. coli cell lysate using a two‐phase aqueous micellar system

10.1002/bit.20806

Crossref journal-article

Wiley

Biotechnology and Bioengineering (311)

Abstract

AbstractGreen fluorescent protein (GFP) has been proposed as an ideal choice for a protein‐based biological indicator for use in the validation of decontamination or disinfection treatments. In this article, we present a potentially scalable and cost‐effective way to purify recombinant GFP, produced by fermentation in Escherichia coli, by affinity‐enhanced extraction in a two‐phase aqueous micellar system. Affinity‐enhanced partitioning, which improves the specificity and yield of the target protein by specific bioaffinity interactions, has been demonstrated. A novel affinity tag, family 9 carbohydrate‐binding module (CBM9) is fused to GFP, and the resulting fusion protein is affinity‐extracted in a decyl β‐D‐glucopyranoside (C10G1) two‐phase aqueous micellar system. In this system, C10G1 acts as phase forming and as affinity surfactant. We will further demonstrate the implementation of this concept to attain partial recovery of affinity‐tagged GFP from a clarified E. coli cell lysate, including the simultaneous removal of other contaminating proteins. The cell lysate was partitioned at three levels of dilution (5×, 10×, and 40×). Irrespective of the dilution level, CBM9‐GFP was found to partition preferentially to the micelle‐rich phase, with the same partition coefficient value as that found in the absence of the cell lysate. The host cell proteins from the cell lysate were found to partition preferentially to the micelle‐poor phase, where they experience less excluded‐volume interactions. The demonstration of proof‐of‐principle of the direct affinity‐enhanced extraction of CBM9‐GFP from the cell lysate represents an important first step towards developing a cost‐effective separation method for GFP, and more generally, for other proteins of interest. © 2006 Wiley Periodicals, Inc.

Bibliography

Mazzola, P. G., Lam, H., Kavoosi, M., Haynes, C. A., Pessoa, A., Penna, T. C. V., Wang, D. I. C., & Blankschtein, D. (2006). Affinityâtagged green fluorescent protein (GFP) extraction from a clarified E. coli cell lysate using a twoâphase aqueous micellar system. Biotechnology and Bioengineering, 93(5), 998â1004. Portico.

Authors 8

Priscila G. Mazzola (first)
Henry Lam (additional)
Mojgan Kavoosi (additional)
Charles A. Haynes (additional)
Adalberto Pessoa (additional)
Thereza Christina Vessoni Penna (additional)
Daniel I.C. Wang (additional)
Daniel Blankschtein (additional)

References 30 Referenced 29

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10.1016/S0308-8146(98)00177-0
ISO 11134.1994.International standard: Sterilization of health care products—Requirements for validation and routine control—Industrial moist heat sterilization.
ISO 14937.2000.International standard: Sterilization of health care products—General requirements for characterization of a sterilizing agent and the development validation and routine control of a sterilization process for medical devices.
10.1016/0304-4165(96)00031-1
10.1002/(SICI)1097-0290(1999)66:4<247::AID-BIT6>3.0.CO;2-5
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Dates

Type	When
Created	19 years, 6 months ago (Feb. 9, 2006, 8:46 p.m.)
Deposited	1 year, 10 months ago (Oct. 18, 2023, 6:10 a.m.)
Indexed	1 year, 2 months ago (June 7, 2024, 1:15 p.m.)
Issued	19 years, 6 months ago (Feb. 9, 2006)
Published	19 years, 6 months ago (Feb. 9, 2006)
Published Online	19 years, 6 months ago (Feb. 9, 2006)
Published Print	19 years, 4 months ago (April 5, 2006)

Funders 0

None

BibTeX

@article{Mazzola_2006, title={Affinity‐tagged green fluorescent protein (GFP) extraction from a clarified E. coli cell lysate using a two‐phase aqueous micellar system}, volume={93}, ISSN={1097-0290}, url={http://dx.doi.org/10.1002/bit.20806}, DOI={10.1002/bit.20806}, number={5}, journal={Biotechnology and Bioengineering}, publisher={Wiley}, author={Mazzola, Priscila G. and Lam, Henry and Kavoosi, Mojgan and Haynes, Charles A. and Pessoa, Adalberto and Penna, Thereza Christina Vessoni and Wang, Daniel I.C. and Blankschtein, Daniel}, year={2006}, month=feb, pages={998–1004} }

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