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. 2008 Nov 7;6(21):3977-82.
doi: 10.1039/b811501j. Epub 2008 Sep 5.

Optimisation of chemical protein cleavage for erythropoietin semi-synthesis using native chemical ligation

Jonathan P Richardson  1 , Derek Macmillan
Affiliations

Optimisation of chemical protein cleavage for erythropoietin semi-synthesis using native chemical ligation

Jonathan P Richardson et al. Org Biomol Chem. .

Abstract

Selective protein cleavage at methionine residues is a useful method for the production of bacterially derived protein fragments containing an N-terminal cysteine residue required for native chemical ligation. Here we describe an optimised procedure for cyanogen bromide-mediated protein cleavage, and ligation of the resulting fragments to afford biologically active proteins.

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Figures

Fig. 1
Fig. 1. (a) CNBr-mediated protein cleavage yielding an N-terminal cysteine. (b) NCL reaction between synthetic EPO (1–28)-SBn thioester and a bacterially-derived fragment visualised using an anti-EPO monoclonal antibody blot which recognises the synthetic fragment, once appended to the bacterial fragment (product mass = 18.5 kDa).
Fig. 2
Fig. 2. Optimisation of CNBr protein cleavage. (a) The His10-fusion protein prior to cleavage; calculated average mass = 17813.3 Da. (b) Isolated protein after CNBr cleavage in 80% formic acid. (c) Expansion of the molecular ion region shows multiple formylation. (d) The same protein sample after treatment with 0.1 M HCl in 6 M guanidine·HCl for 16 h, calculated average mass = 15292.7 Da (an identical spectrum is obtained upon exposure of the sample to 0.1 M NaOH in 6 M guanidine·HCl for 30 min). (e) Protein sample after treatment with 0.1 M NaOH in 6 M guanidine·HCl for 16 h.
Scheme 1
Scheme 1. Synthesis of EPO residues 1–28 as a C-terminal benzyl thioester. Amino acid 1 was introduced at position 24. Reagents and conditions: (a) HBTU–HOBt–DIPEA (b) ICH2CN, DIPEA, DMF, 24 h (c) BnSH, NaSPh, DMF, 16 h then 95 : 2.5 : 2.5 v/v/v TFA–EDT–H2O, 5 h.
Fig. 3
Fig. 3. NCL between synthetic thioester 2 and recombinant EPO (residues 29–166). The mass spectrum (left) is observed for the ligation product after 4 h and corresponds to the desired mass (right). Reagents and conditions: (a) 6 M guanidine·HCl, 300 mM Na phosphate buffer; pH 7.5, 50 mM MPAA, 20 mM TCEP, 4 h. (b) 2% w/v N-lauroylsarcosine, 50 mM Tris·HCl; pH 8.0, 40 μM CuSO4. The schematic EPO structure is modified from PDB entry 1BUY.
Fig. 4
Fig. 4. (a) Receptor binding assay comparing the glycosylated rhEPO standard (squares) and the bacterially-derived EPO[M54L] (circles). (b) TF-1 cell proliferation assay for bacterially-derived EPO[M54L]. (c) TF-1 cell proliferation assay for semi-synthetic EPO prepared by NCL.
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