Fusion tails for the recovery and purification of recombinant proteins

Ford, Clark; Suominen, Ilari; Glatz, Charles E.

doi:10.1016/1046-5928(91)90057-p

Cited by 144 publications

(84 citation statements)

References 81 publications

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“…Human embryonic kidney cells (293 cells) were transfected with vectors containing the wild-type HLA-H cDNA or the cDNA with either the C282Y or H63D mutation. The FLAG octapeptide sequence was fused onto the carboxyl terminus of each, providing a specific tag for detection of the expressed proteins (13). We established individual stable cell lines expressing the three proteins.…”

Section: Resultsmentioning

confidence: 99%

The Hemochromatosis Founder Mutation in HLA-H Disrupts β2-Microglobulin Interaction and Cell Surface Expression

Feder¹,

Tsuchihashi²,

Irrinki³

et al. 1997

Journal of Biological Chemistry

450

304

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We recently reported the positional cloning of a candidate gene for hereditary hemochromatosis (HH), called HLA-H, which is a novel member of the major histocompatibility complex class I family. A mutation in this gene, cysteine 282 3 tyrosine (C282Y), was found to be present in 83% of HH patient DNAs, while a second variant, histidine 63 3 aspartate (H63D), was enriched in patients heterozygous for C282Y. The functional relevance of either mutation has not been described. Coimmunoprecipitation studies of cell lysates from human embryonic kidney cells transfected with wild-type or mutant HLA-H cDNA demonstrate that wild-type HLA-H binds ␤ 2 -microglobulin and that the C282Y mutation, but not the H63D mutation, completely abrogates this interaction. Immunofluorescence labeling and subcellular fractionations demonstrate that while the wild-type and H63D HLA-H proteins are expressed on the cell surface, the C282Y mutant protein is localized exclusively intracellularly. This report describes the first functional significance of the C282Y mutation by suggesting that an abnormality in protein trafficking and/or cell-surface expression of HLA-H leads to HH disease.Hereditary hemochromatosis (HH) 1 is an autosomal recessive disorder of iron metabolism and represents one of the most common inherited disorders in individuals of Northern European descent with an estimated carrier frequency between 1 in 8 and 1 in 10 (1, (2). In patients with HH, excessive iron deposition in a variety of organs leads to multi-organ dysfunction. Recently, we reported a mutation in a novel MHC class I-like gene, called HLA-H (3). Eighty-three percent of HH patient DNAs were found to be homozygous for this mutation, which consists of a single base transition of G to A and results in a change of cysteine 282 3 tyrosine (C282Y). Subsequent reports have confirmed the high frequency of this founder mutation in other HH patients (4 -6), providing further support that HLA-H is the primary HH locus. A second missense mutation, histidine 63 3 aspartate (H63D), was also reported that was enriched in heterozygotes with the C282Y mutation (eight of nine cases) (3). The specific role that either of these mutations in HLA-H play in the etiology of HH disease has not been elucidated.The HLA-H protein is similar to MHC class I family molecules including HLA-A2, nonclassical class I molecules such as HLA-G, and the human neonatal Fc receptor (FcRn). All four of the invariant cysteine residues that form disulfide bridges in the ␣ 2 and ␣ 3 domains of MHC class I family members are present in the HLA-H protein. One of these conserved cysteine residues is altered in the C282Y mutation. The integrity of the conserved disulfide linkages has been suggested to be critical for proper maintenance of the secondary and tertiary structure of the protein allowing interactions with accessory molecules such as ␤ 2 -microglobulin (7). Importantly, the functional significance of an interaction between ␤ 2 -microglobulin and an unknown class I-like molecule in HH disease was s...

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Section: Resultsmentioning

confidence: 99%

The Hemochromatosis Founder Mutation in HLA-H Disrupts β2-Microglobulin Interaction and Cell Surface Expression

Feder¹,

Tsuchihashi²,

Irrinki³

et al. 1997

Journal of Biological Chemistry

450

304

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“…Tandem affinity purification is an approach where the protein of interest is fused in-frame to two affinity tags, generally separated by a protease cleavage site. The original TAP tag consisted of a calmodulin binding peptide, a TEV cleavage site, followed by two immunoglobulin binding domains of Staphylococcus aureus protein A (ProtA) [158] and demonstrated maximum protein recovery compared to alternative affinity tags such as FLAG-, Strep-, His-, calmodulin-binding protein-, and cellulose-binding domain-tags [162,163]. TAP tagging involves two sequential purification steps as illustrated in Fig.…”

Section: Tandem Affinity Purification and Dual-tagging Methodsmentioning

confidence: 99%

Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications

Young

Britton

Robinson

2012

Biotechnology Journal

410

267

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Protein fusion tags are indispensible tools used to improve recombinant protein expression yields, enable protein purification, and accelerate the characterization of protein structure and function. Solubility-enhancing tags, genetically engineered epitopes, and recombinant endoproteases have resulted in a versatile array of combinatorial elements that facilitate protein detection and purification in microbial hosts. In this comprehensive review, we evaluate the most frequently used solubility-enhancing and affinity tags. Furthermore, we provide summaries of well-characterized purification strategies that have been used to increase product yields and have widespread application in many areas of biotechnology including drug discovery, therapeutics, and pharmacology. This review serves as an excellent literature reference for those working on protein fusion tags.

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“…Thus trypsin, like factor X,, may be unable to access the Arg residue of the spacer in the native fusion protein. Unfortunately, no method currently exists for the precise liberation of an intact target protein from its carrier (Ford et al, 1991;Nilsson et al, 1992). In addition to those described above, two other properties of S-peptide increase its versatility as a carrier in fusion proteins.…”

Section: Discussionmentioning

confidence: 99%

“…Fortunately, these processes can be generalized by using recombinant DNA technology to produce fusion proteins in which target proteins are fused to carrier polypeptides (UhlCn & Moks, 1990; Ford et al, 1991;Nilsson et al, 1992). The affinity of the carrier for a specific ligand can enable the facile detection, immobilization, and purification, of a fusion protein.…”

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confidence: 99%

Ribonuclease S‐peptide as a carrier in fusion proteins

1993

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S-peptide (residues [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] and S-protein (residues are the enzymatically inactive products of the limited digestion of ribonuclease A by subtilisin. S-peptide binds S-protein with high affinity to form ribonuclease S, which has full enzymatic activity. Recombinant DNA technology was used to produce a fusion protein having three parts: carrier, spacer, and target. The two carriers used were the first 15 residues of S-peptide (S15) and a mutant S15 in which Asp 14 had been changed to Asn (D14N S15). The spacer consisted of three proline residues and a fourresidue sequence recognized by factor X, protease. The target was /3-galactosidase. The interaction between the S-peptide portion of the fusion protein and immobilized S-protein allowed for affinity purification of the fusion protein under denaturing (S15 as carrier) or nondenaturing (D14N S15 as carrier) conditions. A sensitive method was developed to detect the fusion protein after sodium dodecyl sulfate-polyacrylamide gel electrophoresis by its ribonuclease activity following activation with S-protein. S-peptide has distinct advantages over existing carriers in fusion proteins in that it combines a small size (215 residues), a tunable affinity for ligand (Kd L lo-' M), and a high sensitivity of detection (~1 0 "~ mol in a gel).

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Fusion tails for the recovery and purification of recombinant proteins

Cited by 144 publications

References 81 publications

The Hemochromatosis Founder Mutation in HLA-H Disrupts β2-Microglobulin Interaction and Cell Surface Expression

The Hemochromatosis Founder Mutation in HLA-H Disrupts β2-Microglobulin Interaction and Cell Surface Expression

Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications

Ribonuclease S‐peptide as a carrier in fusion proteins

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