Biotinylated-sortase self-cleavage purification BISOP method for cell-free produced proteinsReportar como inadecuado




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BMC Biotechnology

, 10:42

First Online: 04 June 2010Received: 10 January 2010Accepted: 04 June 2010

Abstract

BackgroundTechnology used for the purification of recombinant proteins is a key issue for the biochemical and structural analyses of proteins. In general, affinity tags, such as glutathione-S-transferase or six-histidines, are used to purify recombinant proteins. Since such affinity tags often interfere negatively with the structural and functional analyses of proteins, they are usually removed by treatment with proteases. Previously, Dr. H. Mao reported self-cleavage purification of a target protein by fusing the sortase protein to its N-terminal end, and subsequently obtained tag-free recombinant protein following expression in Escherichia coli. This method, however, is yet to be applied to the cell-free based protein production.

ResultsThe histidine tag-based self-cleavage method for purifying proteins produced by the wheat cell-free protein synthesis system showed high background, low recovery, and unexpected cleavage between the N-terminally fused sortase and target protein during the protein synthesis. Addition of calcium chelator BAPTA to the cell-free reaction inhibited the cleavage. In order to adapt the sortase-based purification method to the cell-free system, we next used biotin as the affinity tag. The biotinylated sortase self-cleavage purification BISOP method provided tag-free, highly purified proteins due to improved recovery of proteins from the resin. The N-terminal sequence analysis of the GFP produced by the BISOP method revealed that the cleavage indeed occurred at the right cleavage site. Using this method, we also successfully purified the E2 heterocomplex of USE2N and USE2v1. The c-terminal src kinase CSK obtained by the BISOP method showed high activity in phosphorylating the Src protein. Furthermore, we demonstrated that this method is suitable for automatically synthesizing and purifying proteins using robots.

ConclusionWe demonstrated that the newly developed BISOP method is very useful for obtaining high quality, tag-free recombinant proteins, produced using the cell-free system, for biochemical and structural analyses.

AbbreviationssrtAsortase SrtA

GFPgreen fluorescent protein

SGKserum-glucocorticoid regulated kinase 1

CSKc-src tyrosine kinase

UBE2Nubiquitin-conjugating enzyme E2N

UBE2v1ubiquitin-conjugating enzyme E2 variant 1

AMPKa15-AMP-activated protein kinase alpha 1 catalytic subunit

PRKAA1protein kinase, AMP-activated: alpha 1 catalytic subunit

MAPK12mitogen-activated protein kinase 12

p38gp38 gamma

Pfs25Plasmodium falciparum 25 kDa ookinete surface antigen precursor

BAPTA 12-biso-aminophenoxyethane-N,N,N-,N-tetraacetic acid

PBSphosphate buffered saline.

Electronic supplementary materialThe online version of this article doi:10.1186-1472-6750-10-42 contains supplementary material, which is available to authorized users.

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Autor: Satoko Matsunaga - Kazuhiro Matsuoka - Kouhei Shimizu - Yaeta Endo - Tatsuya Sawasaki

Fuente: https://link.springer.com/article/10.1186/1472-6750-10-42







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