Intein-mediated one-step purification of Escherichia coli secreted human antibody fragments

Wu, Wentao; Miller, Keith; Coolbaugh, Michael J.; Wood, David

doi:10.1016/j.pep.2010.12.004

Cited by 19 publications

(10 citation statements)

References 38 publications

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“…This work clearly demonstrates the advantages of the pH and temperature control of our intein, which allows efficient cleaving to take place without interfering with the native disulfide bonds of the targets. This work has been recently published in Protein Expression and Purification 27 . At laboratory scale, we combined advanced recombination-based cloning methods with intein-based purification methods, to produced purified ELP-intein tagged targets in high cell-density E. coli fermentation.…”

Section: Brief Summary Of Results From Princeton Workmentioning

confidence: 99%

“…This method produces extremely high cell densities of E. coli, and provides efficient expression of uncleaved precursor proteins for purification. A single round of optimization produced highly active and pure OPH at a yield of close to 100 mg per liter, without any conventional chromatographic processes ( Figure 6) 27 , and on high cell-density fermentations with products expressed in E. coli 5 . Brief Summary of the Results from the Current Grant (at OSU) Fermentation Development.…”

Section: Brief Summary Of Results From Princeton Workmentioning

confidence: 99%

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Production of Self-Purifying Proteins in a Variety of Expression Hosts with Focus on Organophosphorus Hydrolase

Wood¹

2012

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The public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information. This project focuses on the development of highly effective non-chromatographic methods for purifying recombinant proteins and enzymes. This report covers work at Ohio State University, which focuses on the further optimization of the purification technology in the PI's new laboratory. A key aspect of the technology involves the use of a self-cleaving protein module, which can be used to make any purification tag self-cleaving. By combining this module with a variety of purification tags, we have generated a number of simple, inexpensive and highly ABSTRACTThis project focuses on the development of highly effective non-chromatographic methods for purifying recombinant proteins and enzymes. This report covers work at Ohio State University, which focuses on the further optimization of the purification technology in the PI's new laboratory. A key aspect of the technology involves the use of a self-cleaving protein module, which can be used to make any purification tag self-cleaving. By combining this module with a variety of purification tags, we have generated a number of simple, inexpensive and highly effective methods for protein purification. We have applied these methods to the production of several proteins, including organophosphohydrolase (OPH), which can be used to degrade nerve agents and environmentally persistent insecticides. We have further demonstrated these methods in high cell-density fermentation at laboratory scale, and have provided evidence of their effectiveness. Our most recent work has been on the optimization of the fermentation process itself, as well as a more biochemical optimization of the expression system. Overall, the ARO support on this project has yielded two patent applications, seven peer-reviewed papers and one book chapter. In addition to these, three manuscripts are in late stages of preparation.

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Section: Brief Summary Of Results From Princeton Workmentioning

confidence: 99%

Section: Brief Summary Of Results From Princeton Workmentioning

confidence: 99%

Production of Self-Purifying Proteins in a Variety of Expression Hosts with Focus on Organophosphorus Hydrolase

Wood¹

2012

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“…Apart from ELPs, the pH-induced inteins were also fused to chitin binding domain (CBD) for standard affinity chromatography purification via intein-mediated cleavage under the same mild conditions. This strategy has been employed for different proteins such as human antibody fragments, E. coli maltose binding protein (Wu et al, 2011) and human epidermal growth factor (Esipov et al, 2008). On the other hand, examples of thiol-induced inteins have been reported in the literature as fusion partners of oleosin for purification and removal of xyalanase (Liu et al, 2008) and hydantoinase (Chiang et al, 2007).…”

Section: Alternative Schemes For Tag Removalmentioning

confidence: 99%

Challenges and opportunities in the purification of recombinant tagged proteins

Pina

Lowe

Roque

2014

Biotechnology Advances

126

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The purification of recombinant proteins by affinity chromatography is one of the most efficient strategies due to the high recovery yields and purity achieved. However, this is dependent on the availability of specific affinity adsorbents for each particular target protein. The diversity of proteins to be purified augments the complexity and number of specific affinity adsorbents needed, and therefore generic platforms for the purification of recombinant proteins are appealing strategies. This justifies why genetically encoded affinity tags became so popular for recombinant protein purification, as these systems only require specific ligands for the capture of the fusion protein through a pre-defined affinity tag tail. There is a wide range of available affinity pairs "tag-ligand" combining biological or structural affinity ligands with the respective binding tags. This review gives a general overview of the well-established "tag-ligand" systems available for fusion protein purification and also explores current unconventional strategies under development.

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“…The protein of interest is released as a thioester of the small molecule which, depending on the intended use, can be used to carry‐out C‐terminal modification or simply hydrolyzed to generate a C‐terminal carboxylate. While this technology has worked well for a large number of proteins from small anti‐bacterial toxins to antibody fragments to human choline acetyltransferase it has not become a widely used method for general protein purification. Rather it has found its widest use to generate proteins that can subsequently be used for C‐terminal modification via the thioester generated during cleavage.…”

Section: Self‐cleaving Affinity Tagsmentioning

confidence: 99%

To fuse or not to fuse: What is your purpose?

et al. 2013

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Since the dawn of time, or at least the dawn of recombinant DNA technology (which for many of today's scientists is the same thing), investigators have been cloning and expressing heterologous proteins in a variety of different cells for a variety of different reasons. These range from cell biological studies looking at protein-protein interactions, post-translational modifications, and regulation, to laboratory-scale production in support of biochemical, biophysical, and structural studies, to large scale production of potential biotherapeutics. In parallel, fusion-tag technology has grown-up to facilitate microscale purification (pull-downs), protein visualization (epitope tags), enhanced expression and solubility (protein partners, e.g., GST, MBP, TRX, and SUMO), and generic purification (e.g., His-tags, streptag, and FLAG TM -tag). Frequently, these latter two goals are combined in a single fusion partner. In this review, we examine the most commonly used fusion methodologies from the perspective of the ultimate use of the tagged protein.That is, what are the most commonly used fusion partners for pull-downs, for structural studies, for production of active proteins, or for large-scale purification? What are the advantages and limitations of each? This review is not meant to be exhaustive and the approach undoubtedly reflects the experiences and interests of the authors. For the sake of brevity, we have largely ignored epitope tags although they receive wide use in cell biology for immunopreciptation.

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Intein-mediated one-step purification of Escherichia coli secreted human antibody fragments

Cited by 19 publications

References 38 publications

Production of Self-Purifying Proteins in a Variety of Expression Hosts with Focus on Organophosphorus Hydrolase

Production of Self-Purifying Proteins in a Variety of Expression Hosts with Focus on Organophosphorus Hydrolase

Challenges and opportunities in the purification of recombinant tagged proteins

To fuse or not to fuse: What is your purpose?

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