Genetic Approach to Facilitate Purification of Recombinant Proteins with a Novel Metal Chelate Adsorbent

Hochuli, Erich; Bannwarth, Willi; Döbeli, Heinz; Gentz, Reiner; Stüber, Dietrich

doi:10.1038/nbt1188-1321

Cited by 976 publications

(680 citation statements)

References 16 publications

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“…Virtually all large structural genomics centers use immobilized metal affinity chromatography (IMAC) as their principal affinity strategy. Ni(II)-nitrilotriacetic acid (Ni-NTA), which exhibits a high affinity for adjacent histidine residues, is the most commonly used matrix for IMAC [27,28]. The His-tag combines the advantages of small size with the added benefit of interacting with a chromatographic matrix (e.g.…”

Section: Enhancing the Solubility Of Recombinant Proteinsmentioning

confidence: 99%

Making the most of affinity tags

Waugh

2005

Trends in Biotechnology

483

324

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Section: Enhancing the Solubility Of Recombinant Proteinsmentioning

confidence: 99%

Making the most of affinity tags

Waugh

2005

Trends in Biotechnology

483

324

View full text Add to dashboard Cite

“…The latter amino acid sequence binds e ciently to metal ions and can be used to isolate the protein using metal chelate chromatography (Hochuli et al, 1988).…”

Section: Construction and Stable Cloning Of Full-length Atm Open Readmentioning

confidence: 99%

Recombinant ATM protein complements the cellular A-T phenotype

et al. 1997

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Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by neurodegeneration, immunode®ciency, cancer predisposition, genome instability and radiation sensitivity. The cellular phenotype of A-T points to defects in signal transduction pathways involved in activation of cell cycle checkpoints by free radical damage, and other pathways that mediate the transmission of speci®c mitogenic stimuli. The product of the responsible gene, ATM, belongs to a family of large proteins that contribute to maintaining genome stability and cell cycle progression in various organisms. A recombinant vector that stably expresses a full-length ATM protein is a valuable tool for its functional analysis. We constructed and cloned a recombinant, full-length open reading frame of ATM using a combination of vectors and hosts that overcame an inherent instability of this sequence. Recombinant ATM was stably expressed in insect cells using a baculovirus vector, albeit at a low level, and in human A-T cells using an episomal expression vector. An amino-terminal FLAG epitope added to the protein allowed highly speci®c detection of the recombinant molecule by immunoblotting, immunoprecipitation and immunostaining, and its isolation using immunoanity. Similar to endogenous ATM, the recombinant protein is located mainly in the nucleus, with low levels in the cytoplasm. Ectopic expression of ATM in A-T cells restored normal sensitivity to ionizing radiation and the radiomimetic drug neocarzinostatin, and a normal pattern of post-irradiation DNA synthesis, which represents an Sphase checkpoint. These observations indicate that the recombinant, epitope-tagged protein is functional. Introduction into this molecule of a known A-T missense mutation, Glu2904Gly, resulted in apparent instability of the protein and inability to complement the A-T phenotype. These ®ndings indicate that the physiological defects characteristic of A-T cells result from the absence of the ATM protein, and that this de®ciency can be corrected by ectopic expression of this protein.

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“…For a direct comparison of the usefulness of two C-terminal tags for protein purification the 6 C-terminal histidine residues of PmSUC2-Bio-His6 were used to enrich PmSUC2-Bio-His6 fi'om S. cerevisiae GDY2133 with a Ni2+-nitrilo acidic acidcolumn (data not shown) [15]. Due to the permanent loss of PmSUC2-Bio-His6 during the various washes and due to the co-purification of an inherent, histidine-rich yeast protein less PmSUC2-Bio-His6 protein of lower purity was obtained.…”

Section: Discussionmentioning

confidence: 99%

“…The full-length PmSUC2 cDNA clone pTPI8 [13] was used as template for a polymerase chain reaction (PCR) with an universal sequencing primer and the oligonucleotide PTP1XH6 (5'-GAA GGA ATT CTA GTG GTG GTG GTG GTG GTG GCT CGA GTG ACC TCC AGC CAC AC-3') to add the sequence Ser-Ser-His-His-His-His-HisHis to the C-terminus of the PmSUC2 protein [15]. The two serine residues were encoded by a unique XhoI restriction site, the last histidine codon was followed by a TAG stop codon and an EcoRI site.…”

Section: Construction Of Pmsuc2-bio-his6mentioning

confidence: 99%

Rapid purification of a functionally active plant sucrose carrier from transgenic yeast using a bacterial biotin acceptor domain

1995

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The only way to obtain a clear picture of the structure of these proteins is the generation of 2-dimensional or 3-dimensional crystals or the structural analysis of purified protein by NMR. For most membrane proteins, however, already the first steps towards any of these techniques are hindered by the low expression levels of these proteins, by their extreme hydrophobicity and the concomitant difficulties during purification.The attachment of hydrophilic tags to such proteins has become an important tool to overcome at least part of these problems [9 12]. The present paper describes the advantages of a bacterial biotin acceptor domain for the one-step purification of the recombinant P major sucrose carrier PmSUC2 [13] from transgenic yeast.

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Genetic Approach to Facilitate Purification of Recombinant Proteins with a Novel Metal Chelate Adsorbent

Cited by 976 publications

References 16 publications

Making the most of affinity tags

Making the most of affinity tags

Recombinant ATM protein complements the cellular A-T phenotype

Rapid purification of a functionally active plant sucrose carrier from transgenic yeast using a bacterial biotin acceptor domain

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