Purification of recombinant green fluorescent protein by three-phase partitioning

Jain, Shuchi; Singh, Rajni; Gupta, Munishwar N.

doi:10.1016/j.chroma.2004.01.007

Cited by 46 publications

(27 citation statements)

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“…While TPP has previously been used for protein concentration and purification (Dennison and Lovrien 1997;Jain et al 2004), in the present study we have applied TPP to successfully refold and purify a variety of recombinant proteins from inclusion bodies, several of which could not be purified by conventional procedures. There is a debate about the exact mechanism of TPP-mediated refolding (Lovrien et al 1995;Dennison and Lovrien 1997;Jain et al 2004;Kiss and Borbas 2003).…”

Section: Discussionmentioning

confidence: 99%

“…There is a debate about the exact mechanism of TPP-mediated refolding (Lovrien et al 1995;Dennison and Lovrien 1997;Jain et al 2004;Kiss and Borbas 2003). However, both ammonium sulfate and alcohols have been reported to be useful additives during refolding (Middelberg 2002).…”

Section: Discussionmentioning

confidence: 99%

“…In the first step, using only 5% (wv À1 ) ammonium sulfate was favorable. The ratio of t-butanol to aqueous phase volume was fixed at 1:1 as this ratio has worked well in the earlier TPP experiments (Dennison and Lovrien 1997;Jain et al 2004). The first TPP, in the case of native RNase A, did not precipitate the protein at the interfacial layer.…”

Section: The General Strategymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…Factorial and other screens for refolding have been developed (Hofmann et al 1995;Armstrong et al 1999;Bajaj et al 2004) to facilitate the search of appropriate refolding conditions. In the present study we describe the first application of the technique of three-phase partitioning (TPP) (Lovrien et al 1995;Dennison and Lovrien 1997;Jain et al 2004;Przybycien et al 2004) to obtain active refolded proteins from solubilized inclusion bodies without any chromatographic steps. We have used TPP to refold 12 different proteins from inclusion bodies, namely, ribonuclease A (RNase A) (Crook et al 1960;Wagner et al 1983;Laity et al 1993;delCardayré et al 1995;Okorokov et al 1995), the first two domains of human CD4 (CD4D12) (Dalgleish et al 1984;Doyle and Strominger 1987;Sharma et al 2005), five insoluble constructs of the protein tyrosine phosphatases (PTPs) from Drosophila melanogaster (Madan and Gopal 2008), and five highly unstable and aggregation prone mutants of the E. coli proteins controller of cell division or death B (CcdB), maltose binding protein (MBP), and thioredoxin (Trx) Bajaj et al 2005;Das et al 2007).…”

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Refolding and simultaneous purification by three‐phase partitioning of recombinant proteins from inclusion bodies

et al. 2008

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Many recombinant eukaryotic proteins tend to form insoluble aggregates called inclusion bodies, especially when expressed in Escherichia coli. We report the first application of the technique of threephase partitioning (TPP) to obtain correctly refolded active proteins from solubilized inclusion bodies. TPP was used for refolding 12 different proteins overexpressed in E. coli. In each case, the protein refolded by TPP gave either higher refolding yield than the earlier reported method or succeeded where earlier efforts have failed. TPP-refolded proteins were characterized and compared to conventionally purified proteins in terms of their spectral characteristics and/or biological activity. The methodology is scaleable and parallelizable and does not require subsequent concentration steps. This approach may serve as a useful complement to existing refolding strategies of diverse proteins from inclusion bodies.Keywords: three-phase partitioning; protein refolding; recombinant ribonuclease A; CcdB mutants; human CD4; inclusion bodies Supplemental material: see www.proteinscience.org Upon expression in Escherichia coli, many recombinant proteins form dense, insoluble aggregates called inclusion bodies (Taylor et al. 1986). It is therefore necessary to develop efficient and scalable procedures for solubilization and refolding of recombinant proteins from inclusion bodies (Misawa and Kumagai 1999;Middelberg 2002).Refolding is typically carried out using either simple dilution into appropriate refolding conditions, dialysis or on a column (Stempfer et al. 1996;Rogl et al. 1998). Finding appropriate refolding conditions is generally the most difficult step of the purification process. Aggregation and precipitation of protein during refolding are commonly encountered problems. Factorial and other screens for refolding have been developed (Hofmann et al. 1995;Armstrong et al. 1999;Bajaj et al. 2004) to facilitate the search of appropriate refolding conditions. In the present study we describe the first application of the technique of three-phase partitioning (TPP) (Lovrien et al. 1995;Dennison and Lovrien 1997;Jain et al. 2004;Przybycien et al. 2004) to obtain active refolded proteins from solubilized inclusion bodies without any chromatographic steps. We have used TPP to refold 12 different proteins from inclusion bodies, namely, ribonuclease A Reprint requests to: Munishwar N. Gupta, Chemistry Department, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110 016, India; e-mail: munishwar48@yahoo.co.uk; fax: 91-011-26581073.Abbreviations: CcdB, controller of cell division or death B; CD4D12, first two domains of human CD4; C m , the denaturant concentration at which one half of the protein molecules are unfolded; GdmCl, guanidium chloride; IPTG, isopropyl b-D-1-thiogalactopyranoside; MBP, maltose binding protein; PTPs, protein tyrosine phosphatases; RNase A, ribonuclease A; SPR, surface plasmon resonance; TPP, three-phase partitioning; Trx, E. coli thioredoxin.Article and publication are at

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: The General Strategymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Refolding and simultaneous purification by three‐phase partitioning of recombinant proteins from inclusion bodies

et al. 2008

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Chemical cross‐linking of a variety of green fluorescent proteins as Förster resonance energy transfer donors for Yukon orange fluorescent protein: A project‐based undergraduate laboratory experience

Marchioretto

Horton

Berstler

et al. 2018

Biochem Molecular Bio Educ

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Förster resonance energy transfer (FRET) is the basis for many techniques used in biomedical research. Due to its wide use in molecular sensing, FRET is commonly introduced in many biology, chemistry, and physics courses. While FRET is of great importance in the biophysical sciences, the complexity and difficulty of constructing FRET experiments has resulted in limited usage in undergraduate laboratory settings. Here, we present a practical undergraduate laboratory experiment for teaching FRET using a diverse set of green‐emitting fluorescent proteins (FPs) as donors for a cross‐linked Yukon orange FP. This laboratory experiment enables students to make the connection of basic lab procedures to real world applications and can be applied to molecular biology, biochemistry, physical chemistry, and biophysical laboratory courses. Published 2018. This article is a U.S. Government work and is in the public domain in the USA., 46(5):516–522, 2018.

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Single‐step purification of the recombinant green fluorescent protein from intact Escherichia coli cells using preparative PAGE

et al. 2009

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Mechanical and non-mechanical breakages of bacterial cells are usually the preliminary steps in intracellular protein purification. In this study, the recombinant green fluorescent protein (GFP) was purified from intact Escherichia coli cells using preparative PAGE. In this purification process, cells disruption step is not needed. The cellular content of E. coli was drifted out electrically from cells and the negatively charged GFP was further electroeluted from polyacrylamide gel column. SEM investigation of the electrophoresed cells revealed substantial structural damage at the cellular level. This integrated purification technique has successfully recovered the intracellular GFP with a yield of 82% and purity of 95%.

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Purification of recombinant green fluorescent protein by three-phase partitioning

Cited by 46 publications

References 13 publications

Refolding and simultaneous purification by three‐phase partitioning of recombinant proteins from inclusion bodies

Refolding and simultaneous purification by three‐phase partitioning of recombinant proteins from inclusion bodies

Chemical cross‐linking of a variety of green fluorescent proteins as Förster resonance energy transfer donors for Yukon orange fluorescent protein: A project‐based undergraduate laboratory experience

Single‐step purification of the recombinant green fluorescent protein from intact Escherichia coli cells using preparative PAGE

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