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Making ends meet: chemically mediated circularization of recombinant proteins.

Cowper B, Craik DJ, Macmillan D - Chembiochem (2013)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University College London, Christopher Ingold Building, 20 Gordon Street, London, WC1H 0AJ, UK.

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Related in: MedlinePlus

Production of KB1. A) SDS-PAGE analysis of Ni2+-affinity-purified Trx-KB1 fusion protein. M: molecular weight markers. Lane 1: whole-cell lysate, lane 2: soluble fraction, lane 3: insoluble fraction, lane 4: column flow-through, lane 5: column wash (5 mm imidazole), lane 6: column wash (20 mm imidazole), lanes 7–11: eluted fractions (40–500 mm imidazole). B) TEV protease digestion of the fusion protein shows accumulation of Trx (released linear KB1 is not visible on the gel). C) Preparative HPLC allows straightforward separation of the released KB1 from Trx. D) Analytical HPLC (lower panel) and MS (upper panels) characterization of purified linear, cyclic (reduced) and folded KB1 samples. E) HPLC coelution experiment: KB1 (upper panel), native KB1 (isolated from O. affinis, middle panel) and a 1:1 mixture of each peptide (lower panel).
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fig01: Production of KB1. A) SDS-PAGE analysis of Ni2+-affinity-purified Trx-KB1 fusion protein. M: molecular weight markers. Lane 1: whole-cell lysate, lane 2: soluble fraction, lane 3: insoluble fraction, lane 4: column flow-through, lane 5: column wash (5 mm imidazole), lane 6: column wash (20 mm imidazole), lanes 7–11: eluted fractions (40–500 mm imidazole). B) TEV protease digestion of the fusion protein shows accumulation of Trx (released linear KB1 is not visible on the gel). C) Preparative HPLC allows straightforward separation of the released KB1 from Trx. D) Analytical HPLC (lower panel) and MS (upper panels) characterization of purified linear, cyclic (reduced) and folded KB1 samples. E) HPLC coelution experiment: KB1 (upper panel), native KB1 (isolated from O. affinis, middle panel) and a 1:1 mixture of each peptide (lower panel).

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).


Making ends meet: chemically mediated circularization of recombinant proteins.

Cowper B, Craik DJ, Macmillan D - Chembiochem (2013)

Production of KB1. A) SDS-PAGE analysis of Ni2+-affinity-purified Trx-KB1 fusion protein. M: molecular weight markers. Lane 1: whole-cell lysate, lane 2: soluble fraction, lane 3: insoluble fraction, lane 4: column flow-through, lane 5: column wash (5 mm imidazole), lane 6: column wash (20 mm imidazole), lanes 7–11: eluted fractions (40–500 mm imidazole). B) TEV protease digestion of the fusion protein shows accumulation of Trx (released linear KB1 is not visible on the gel). C) Preparative HPLC allows straightforward separation of the released KB1 from Trx. D) Analytical HPLC (lower panel) and MS (upper panels) characterization of purified linear, cyclic (reduced) and folded KB1 samples. E) HPLC coelution experiment: KB1 (upper panel), native KB1 (isolated from O. affinis, middle panel) and a 1:1 mixture of each peptide (lower panel).
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Related In: Results  -  Collection

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fig01: Production of KB1. A) SDS-PAGE analysis of Ni2+-affinity-purified Trx-KB1 fusion protein. M: molecular weight markers. Lane 1: whole-cell lysate, lane 2: soluble fraction, lane 3: insoluble fraction, lane 4: column flow-through, lane 5: column wash (5 mm imidazole), lane 6: column wash (20 mm imidazole), lanes 7–11: eluted fractions (40–500 mm imidazole). B) TEV protease digestion of the fusion protein shows accumulation of Trx (released linear KB1 is not visible on the gel). C) Preparative HPLC allows straightforward separation of the released KB1 from Trx. D) Analytical HPLC (lower panel) and MS (upper panels) characterization of purified linear, cyclic (reduced) and folded KB1 samples. E) HPLC coelution experiment: KB1 (upper panel), native KB1 (isolated from O. affinis, middle panel) and a 1:1 mixture of each peptide (lower panel).
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).

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University College London, Christopher Ingold Building, 20 Gordon Street, London, WC1H 0AJ, UK.

Show MeSH
Related in: MedlinePlus