Protein synthesis by native chemical ligation: expanded scope by using straightforward methodology

Abstract

The total chemical synthesis of proteins has great potential for increasing our understanding of the molecular basis of protein function. The introduction of native chemical ligation techniques to join unprotected peptides next to a cysteine residue has greatly facilitated the synthesis of proteins of moderate size. Here, we describe a straightforward methodology that has enabled us to rapidly analyze the compatibility of the native chemical ligation strategy for X-Cys ligation sites, where X is any of the 20 naturally occurring amino acids. The simplified methodology avoids the necessity of specific amino acid thioester linkers or alkylation of C-terminal thioacid peptides. Experiments using matrix-assisted laser-desorption ionization MS analysis of combinatorial ligations of LYRAX-C-terminal thioester peptides to the peptide CRANK show that all 20 amino acids are suitable for ligation, with Val, Ile, and Pro representing less favorable choices because of slow ligation rates. To illustrate the method's utility, two 124-aa proteins were manually synthesized by using a three-step, four-piece ligation to yield a fully active human secretory phospholipase A(2) and a catalytically inactive analog. The combination of flexibility in design with general access because of simplified methodology broadens the applicability and versatility of chemical protein synthesis.

Figure 1

Parallel native chemical ligation model study of LYRAX-to-CRANK peptide ligations, with X representing all 20 natural amino acids. (A) Under experimental conditions during native chemical ligation, LYRAX–MPAL-activated thioester peptides undergo thioester exchange reactions with benzylmercaptan and thiophenol. (B) Native chemical ligation. COSR of LYRAX–MPAL undergoes nucleophilic attack by the side chain of the N-terminal cysteine residue of CRANK, after which a rapid intramolecular rearrangement produces a native peptide bond at the site of ligation. (C) Simultaneous MALDI-MS readout of combinatorial ligations of crude LYRAX–MPAL-to-CRANK ligations featuring A, V, I, M, and F as C-terminal amino acid-activated thioester residues. (D) Determination of ligation product (filled symbols) and benzylmercaptan–thioester exchange intermediates (open symbols) as a function of time for all 20 LYRAX–MPAL C-terminal activated thioesters. C-terminal amino acids are divided into four groups, in which ligations were completed within 4, 9, or 24 hr or in 48 hr or more, respectively. Left, (t ≤ 4 hr), ∗ indicates the observed LYRAH–CRANK model-peptide ligation rate when monitored by using HPLC analysis.

Figure 2

Protein synthesis by multistep ligation: hsPLAA₂. (A) Sequence of the four peptides comprising the 124-aa polypeptide chain of hsPLA₂. Ligation sites include His(Dnp)–Cys, Gly–Cys, and Leu–Cys. In a parallel synthesis, the underlined His-47 active-site residue was replaced by the isosteric Bta to obtain a chemical hsPLA₂ variant. (B) Synthetic scheme leading to the 124-aa hsPLA₂ polypeptide chain. Typically, unprotected purified peptides were dissolved at 10 mg/ml in 0.1 M phosphate buffer containing 6 M guanidine and 4% benzylmercaptan and thiophenol reaching pH ≈ 7 (1). To avoid polymerization reactions, N-terminal cysteine residues of the activated thioester peptides were protected with Msc groups. After ligation, a 5-min treatment at pH 13 removed N-terminal Msc (2). HPLC yielded the purified ligation product. (3). (C) Electrospray ionization-MS of the reduced polypeptide chain of hsPLA₂–Bta-47 (Upper) and the oxidized refolded enzyme variant (Lower). Mass reconstructions from the m/z ratios show a mass decrease from 13,935 Da to 13,921 Da representing the loss of 14 protons in the formation of seven internal disulfide bonds.