Making ends meet: chemically mediated circularization of recombinant proteins.
Bottom Line: A selective N→S acyl transfer reaction facilitates semi-synthesis of the plant cyclotide kalata B1 from a linear precursor peptide of bacterial origin, through simple appendage of N-terminal cysteine and a thiol-labile C-terminal Gly-Cys motif.This constitutes the first synthesis of a ribosomally derived circular miniprotein, without recourse to protein splicing elements.
Affiliation: Department of Chemistry, University College London, Christopher Ingold Building, 20 Gordon Street, London, WC1H 0AJ, UK.Show MeSH
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Mentions: Bacterial expression of a recombinant kalata B1 linear precursor peptide was facilitated through fusion with an N-terminal thioredoxin (Trx) tag, yielding approximately 60 mg of purified protein per litre of cell culture. Conveniently, one of the six cysteines of wild-type KB1 is preceded by glycine. These sequential glycine and cysteine residues were therefore designated as the respective C- and N-terminal KB1 residues in the genetically encoded sequence, and a seventh cysteine was appended to the C terminus to provide a labile Gly-Cys site for N→S acyl shift. The His-tagged Trx-KB1 fusion protein was purified through immobilized Ni2+-affinity chromatography (Figure 1 A and Figure S1 in the Supporting Information), and the linear KB1 peptide was liberated initially through factor Xa protease (Figures S2 and S3), yielding linear KB1 with an N-terminal cysteine. Subsequently more efficient liberation was achieved by employing tobacco etch virus (TEV) protease (Figure 1 B). The desired KB1 peptide was purified through reversed phase-high performance liquid chromatography (RP-HPLC) in an unoptimised yield of 35 %, based on the Trx-fusion precursor (Figure 1 C), and characterized by mass spectrometry (Figure 1 D, left).
Affiliation: Department of Chemistry, University College London, Christopher Ingold Building, 20 Gordon Street, London, WC1H 0AJ, UK.