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.
Highlights
Technology used for the purification of recombinant proteins is a key issue for the biochemical and structural analyses of proteins
Cleavage of Pfs25 (S25 lane in Figure 1B) and green fluorescent protein (GFP) (Figure 1C) during the protein synthesis process were very low, whereas almost 40% of the synthesized SGK495 protein was cleaved during the cell-free synthesis
By comparing the results of the BAPTA-concentration dependent blocking of the sortase SrtA (srtA) activity and inhibition of the protein synthesis, we concluded that 1 mM BAPTA is optimal for the cell-free synthesis of srtAfusion proteins, because at this concentration of BAPTA the recovery of all full-length srtA fusion proteins improved by approximately 10-20% without any major inhibition of the protein synthesis
Summary
Technology used for the purification of recombinant proteins is a key issue for the biochemical and structural analyses of proteins. Mao reported selfcleavage 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, is yet to be applied to the cell-free based protein production. The purified target protein eluted off the affinity column has only an extra Gly residue on the N-terminus end This excellent approach was designed for the purification of recombinant proteins expressed in Escherichia coli cells. Inability to suppress the srtA activity during the expression of the fused recombinant protein in E. coli was, a major limiting factor for using this otherwise excellent approach as a general tool for the production and purification of recombinant proteins
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