Self-Cleavable Stimulus Responsive Tags for Protein Purification without Chromatography

Ge, Xin; Yang, Dan; Trabbic‐Carlson, Kimberly; Kim, Bumjoon J.; Chilkoti, Ashutosh; Filipe, Carlos D. M.

doi:10.1021/ja0531125

Cited by 97 publications

(83 citation statements)

References 11 publications

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“…The Escherichia coli RNase H1 gene was amplified from E. coli genomic DNA and cloned into the pTME plasmid (a kind gift from Carlos D. M. Filipe, McMaster University, Canada). The purification was done according to the previously published method (8), and the purified product was stored at a stock concentration of 2 mg/ml. The purified RNase H was carefully checked for even low levels of DNase or RNase activity and found to contain no exo-or endonucleolytic activity.…”

Section: Methodsmentioning

confidence: 99%

Sequence Dependence of Chromosomal R-Loops at the Immunoglobulin Heavy-Chain Sμ Class Switch Region

Huang¹,

Yu²,

Balter

et al. 2007

Molecular and Cellular Biology

101

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The mechanism by which the cytidine deaminase activation-induced deaminase (AID) acts at immunoglobulin heavy-chain class switch regions during mammalian class switch recombination (CSR) remains unclear. R-loops have been proposed as a basis for this targeting. Here, we show that the difference between various forms of the S locus that can or cannot undergo CSR correlates well with the locations and detectability of R-loops. The S R-loops can initiate hundreds of base pairs upstream of the core repeat switch regions, and the area where the R-loops initiate corresponds to the zone where the AID mutation frequency begins to rise, despite a constant density of WRC sites in this region. The frequency of R-loops is 1 in 25 alleles, regardless of the presence of the core S repeats, again consistent with the initiation of most R-loops upstream of the core repeats. These findings explain the surprisingly high levels of residual CSR in B cells from mice lacking the core S repeats but the marked reduction in CSR in mice with deletions of the region upstream of the core S repeats. These studies also provide the first analysis of how R-loop formation in the eukaryotic chromosome depends on the DNA sequence.Mammalian immunoglobulin (Ig) genes undergo two types of DNA recombination, in addition to a somatic hypermutation (SHM) process. V(D)J recombination occurs in early lymphocytes and assembles the variable-domain exon so that IgM can be made. Class switch recombination (CSR) occurs only at the Ig heavy-chain locus and is responsible for the change in the heavy-chain isotype from IgM to IgG, IgA, or IgE; this process is also called the heavy-chain isotype switch (6,17,29). CSR occurs at repetitive DNA elements called switch regions, which vary in sequence and length. All of the switch regions are more than 1 kb in length and consist of unit repeats of 25 to 80 bp. All are located downstream of a sterile transcript promoter, which is necessary for CSR. All have a G-rich nontemplate strand, and all are rich in sites at which a cytidine deaminase called activation-induced deaminase (AID) prefers to act, namely, WRC sites (37). The regional nature of CSR, which gave rise to the term regionally specific recombination (15), contrasts with the vast majority of other physiologic recombination systems in biology, which are regarded as site specific. The special features of switch regions (such as being long and repetitive and located downstream of a promoter, having Grich nontemplate strands, and not having sequences conserved among the different switch regions or among vertebrates that carry out CSR) suggested that the mechanism would be unusual relative to other recombination processes in biology.Investigators at the Honjo laboratory discovered the key lymphoid-specific enzyme for both CSR and SHM, AID (22,23). AID is a 26-kDa protein which deaminates C in DNA (5, 25) but only when that DNA is single stranded (2,26,36,38). A key question for CSR and SHM concerns how the DNA becomes single stranded. Because transcription appears to...

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Section: Methodsmentioning

confidence: 99%

Sequence Dependence of Chromosomal R-Loops at the Immunoglobulin Heavy-Chain Sμ Class Switch Region

Huang¹,

Yu²,

Balter

et al. 2007

Molecular and Cellular Biology

101

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“…Furthermore, the ELPs potentially offer several advantages over PNIPAM such as biocompatibility and biodegradablility characters. Based on such interesting properties, ELPs have already been employed as useful molecular tools for biological applications such as protein purification, drug delivery and artificial ECM [17][18][19][20][21][22][23][24][25][26]. In most of these studies, genetically engineered ELPs with high molecular weights were used on the subjects.…”

Section: Introductionmentioning

confidence: 99%

Fabrication of Thermo-Responsive Molecular Layers from Self-Assembling Elastin-Like Oligopeptides Containing Cell-Binding Domain for Tissue Engineering

et al. 2015

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Novel thermo-responsive elastin-like oligopeptides containing cell-binding epitope (Arg-Gly-Asp-Ser sequence); arginine-glycine-aspartic acid-serine (RGDS)-elastin-like peptides (ELP) and RGDS-deg-ELP; were newly prepared as building blocks of self-assembled molecular layer for artificial extra cellular matrix. A detailed analysis of the conformation of the oligo(ELP)s in water and their self-assembling behavior onto hydrophobic surfaces were performed by using circular dichroism, Fourier transform infrared spectroscopy (FTIR), atomic force microscopy and water contact angle measurements. The experimental results revealed that both oligo(ELP)s self-assembled onto hydrophobic surfaces and formed molecular layers based on their thermo-responsive conformational change from hydrous random coil to dehydrated β-turn structure. Effective cell adhesion and spreading behaviors were observed on these self-assembled oligo(ELP) layers. In addition, attached cells were found to be recovered successfully as a cell-sheet by temperature-induced disassembly of oligo(ELP) layer. This achievement provides an important insight to construct novel oligopeptide-based nano-surfaces for the design of smart artificial extra-cellular matrix. OPEN ACCESSPolymers 2015, 7 135

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“…In ITC, an ELP fusion protein is selectively separated from other contaminating biomolecules in cell lysate by the sequential and repeated steps of aggregation, centrifugation, and resolubilization of the fusion protein [10,11]. A number of different proteins have been purified by this method using either centrifugation [11][12][13][14][15][16][17][18] or micro-filtration [19]. The direct purification of ELPs has also been extended to the capture of native proteins by ELP-tagged capture reagents [14][15][16][17].…”

mentioning

confidence: 99%

Purification of recombinant proteins from Escherichia coli at low expression levels by inverse transition cycling

Christensen

Trabbic‐Carlson

Liu

et al. 2007

Analytical Biochemistry

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A crucial and challenging problem in proteomics is purification, identification, and characterization of proteins, some of which are expressed at very low levels. The preferred method for purification of low abundance proteins exploits multiple affinity purification tags on a single recombinant protein e.g. tandem affinity purification (TAP) [1]. However TAP is both experimentally lengthy, involving many sequential binding, washing and elution steps and costly, requiring two different and expensive resins to recover the purified recombinant protein.Hence processing large amounts of cell lysate makes it prohibitively expensive especially for scale-up.Here we present a new and simple strategy to purify soluble recombinant proteins from E. coli at a protein concentration that approaches the limit of a single protein molecule per cell. This method utilizes the unique aggregation properties of elastin-like polypeptides (ELPs) to capture recombinant fusion proteins composed of a target protein and an ELP tag from cell lysate. ELPs are artificial, genetically encodable polypeptides composed the repeating pentapeptides sequence VPGXG, where the guest residue (X) can be any naturally occurring amino acid except Pro [2,3]. ELPs exhibit a unique reversible inverse phase transition behavior; below a critical transition temperature (T t ) ELPs are highly soluble in aqueous solution, however at temperatures even a few degrees Celsius above T t , ELP will undergo a solubilityinsolubility phase transition, leading to aggregation of the polypeptide [4]. The T t of an ELP is a function of a number of variables including identity and stoichiometry of the guest residue, molecular weight, ELP and salt concentration in aqueous solution [5][6][7][8][9].The environmental sensitivity and reversible solubility of ELPs are retained when expressed as recombinant fusions with proteins. This feature can be exploited for non-chromatographic purification of recombinant proteins by inverse transition cycling (ITC) [10]. In ITC, an ELP fusion protein is selectively separated from other contaminating biomolecules in cell lysate by the sequential and repeated steps of aggregation, centrifugation, and resolubilization of the fusion protein [10,11]. A number of different proteins have been purified by this method using either centrifugation [11][12][13][14][15][16][17][18] or micro-filtration [19]. The direct purification of ELPs has also been extended to the capture of native proteins by ELP-tagged capture reagents [14][15][16][17].Proteins expressed at the level of micrograms per liter of culture are difficult to purify by chromatography because of the losses associated with non-specific and irreversible adsorption of the target protein to chromatography resins as well as the relative increase in contamination from host proteins that are non-specifically adsorbed to the chromatography resin are subsequently eluted with the target protein. We demonstrate a new variant of ITC that solves *Corresponding Author. Phone: 919-660-5373, Fax: 919-660-5409,...

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Self-Cleavable Stimulus Responsive Tags for Protein Purification without Chromatography

Cited by 97 publications

References 11 publications

Sequence Dependence of Chromosomal R-Loops at the Immunoglobulin Heavy-Chain Sμ Class Switch Region

Sequence Dependence of Chromosomal R-Loops at the Immunoglobulin Heavy-Chain Sμ Class Switch Region

Fabrication of Thermo-Responsive Molecular Layers from Self-Assembling Elastin-Like Oligopeptides Containing Cell-Binding Domain for Tissue Engineering

Purification of recombinant proteins from Escherichia coli at low expression levels by inverse transition cycling

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