Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element

Chong, Shaorong; Mersha, Fana B.; Comb, Donald G.; Scott, Melissa E.; Landry, David; Vence, Luis M.; Perler, Francine B.; Benner, Jack S.; Kucera, Rebecca; Hirvonen, Christine A.; Pelletier, John; Paulus, Henry; Xu, Min

doi:10.1016/s0378-1119(97)00105-4

Cited by 580 publications

(444 citation statements)

References 29 publications

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“…Inteins are cleaved from the precursor proteins with the concomitant joining of the intein-flanking polypeptides (the exteins) to form a functional protein (Paulus, 2000). Due to the controllable autocatalytic trait of the inteins, they are now used as fusion partners for recombinant protein expression (Chong et al, 1997;Chong et al, 1998). The advantage is that it is possible to use the intein in affinity purification and then through a controlled protein cleavage obtain the protein of interest in a native form without any extraneous amino acid sequences.…”

Section: Recombinant Protein Expression Using Self-splicing Inteinsmentioning

confidence: 99%

Gelatinase-mediated migration and invasion of cancer cells

Björklund

Koivunen

2005

Biochimica et Biophysica Acta (BBA) - Reviews on Cancer

463

609

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Section: Recombinant Protein Expression Using Self-splicing Inteinsmentioning

confidence: 99%

Gelatinase-mediated migration and invasion of cancer cells

Björklund

Koivunen

2005

Biochimica et Biophysica Acta (BBA) - Reviews on Cancer

463

609

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“…Residues 1-353, folded into a native state, are made via recombinant DNA technology and are ligated to residues 354-375, made by solid-phase peptide synthesis (SPPS). The majority of R2 (residues 1-353) is fused to an intein mutant, which lacks the C-extein Asn and Cys residues but contains a purification tag, the chitin-binding domain (CBD) 26 . After recombinant expression, the engineered intein-fusion product catalyzes the first step of the self-cleavage reaction, but not the ensuing splicing steps.…”

Section: Semisynthesis Of R2 Using Eplmentioning

confidence: 99%

Site-specific incorporation of fluorotyrosines into the R2 subunit of E. coli ribonucleotide reductase by expressed protein ligation

2007

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Expressed protein ligation (EPL) allows semisynthesis of a target protein with site-specific incorporation of probes or unnatural amino acids at its N or C termini. Here, we describe the protocol that our lab has developed for incorporating fluorotyrosines (F n Ys) at residue 356 of the small subunit of Escherichia coli ribonucleotide reductase using EPL. In this procedure, the majority of the protein (residues 1-353 out of 375) is fused to an intein domain and prepared by recombinant expression, yielding the protein in a thioester-activated, truncated form. The remainder of the protein, a 22-mer peptide, is prepared by solid-phase peptide synthesis and contains the F n Y at the desired position. Ligation of the 22-mer peptide to the thioester-activated R2 and subsequent purification yield full-length R2 with the F n Y at residue 356. The procedure to generate 100 mg quantities of Y 356 F n Y-R2 takes 3-4 months. INTRODUCTION Problem under investigationThe Escherichia coli ribonucleotide reductase (RNR) catalyzes the conversion of all four nucleotides to their corresponding 2¢-deoxynucleotides and consists of two homodimeric subunits: R1 and R2 (refs. 1-3). R1 is the homodimeric subunit where nucleotide reduction occurs. It contains binding sites for nucleoside diphosphate substrates as well as for deoxynucleoside triphosphate and ATP allosteric effectors, which govern substrate specificity and turnover rates. R2 is the homodimeric subunit, which houses the diiron-tyrosyl radical cofactor (Y 122 ) essential for catalysis. Each turnover requires radical migration from the Y 122 site on R2 to the active site of R1, over a distance of 35 Å . The mechanism of this long-range radical propagation event is an active area of research 4,5 .Structures of active RNR, a 1:1 complex of R1 and R2, are not available. However, Uhlin and Eklund 6 have generated a docking model of this complex from the individual structures of R1 and R2 (refs. 7,8

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“…[51][52] N-Extein Elucidations of the protein splicing mechanism have directed the design of engineered inteins that perform single splice-and-junction cleavage under specific conditions. [53] These inteins, when fused to a particular protein either at its C or N terminus, may lead to the generation of a reactive C-terminal thioester or an N-terminal cysteine, respectively. In the case of the thioester formation (Figure 9), the strategy utilizes a mutation of the C-extein that prevents the splicing reaction to proceed after the initial acyl transfer reaction.…”

Section: Expressed Protein Ligationmentioning

confidence: 99%