Chemical Cleavage of an Asp-Cys Sequence Allows Efficient Production of Recombinant Peptides with an N-Terminal Cysteine Residue

Abstract

Peptides with an N-terminal cysteine residue allow site-specific modification of proteins and peptides and chemical synthesis of proteins. They have been widely used to develop new strategies for imaging, drug discovery, diagnostics, and chip technologies. Here we present a method to produce recombinant peptides with an N-terminal cysteine residue as a convenient alternative to chemical synthesis. The method is based on the release of the desired peptide from a recombinant fusion protein by mild acid hydrolysis of an Asp-Cys sequence. To test the general validity of the method we prepared four fusion proteins bearing three different peptides (20-37 amino acid long) at the C-terminus of a ketosteroid isomerase-derived and two Onconase-derived carriers for the production of toxic peptides in E. coli. The chosen peptides were (C)GKY20, an antimicrobial peptide from the C-terminus of human thrombin, (C)ApoB, an antimicrobial peptide from an inner region of human Apolipoprotein B, and (C)p53pAnt, an anticancer peptide containing the C-terminal region of the p53 protein fused to the cell penetrating peptide Penetratin. Cleavage efficiency of Asp-Cys bonds in the four fusion proteins was studied as a function of pH, temperature, and incubation time. In spite of the differences in the amino acid sequence (GTGDCGKY, GTGDCHVA, GSGTDCGSR, SQGSDCGSR) we obtained for all the proteins a cleavage efficiency of about 70-80% after 24 h incubation at 60 °C and pH 2. All the peptides were produced with very good yield (5-16 mg/L of LB cultures), high purity (>96%), and the expected content of free thiol groups (1 mol per mole of peptide). Furthermore, (C)GKY20 was modified with PyMPO-maleimide, a commercially available fluorophore bearing a thiol reactive group, and with 6-hydroxy-2-cyanobenzothiazole, a reagent specific for N-terminal cysteines, with yields of 100% thus demonstrating that our method is very well suited for the production of fully reactive peptides with an N-terminal cysteine residue.

Use of an asparaginyl endopeptidase for chemo-enzymatic peptide and protein labeling

“…32,33,70 On the other hand, FPBA is a commercially available reagent that offers a fast, selective and simple method for modication, 4,53,54 but preparation of proteins with a free N-terminal cysteine is not universally trivial, as it is prone to side reaction (oxidation and thiazolidinone formation). 30,[55][56][57][58][59] However, when the AEP catalysis and FPBA bioconjugation are combined together, a system that enables both N-and C-terminal ligation with the use of stable labeling agents is developed. 32 Provided that the AEP-ligation/FPBA-coupling method lowers the ratio of label to protein substrate, it is particularly applicable when expensive or non-commercially available labels are used.…”

“…53,54 Nevertheless, preparing proteins with a N-terminal cysteine is case-dependent and can be a challenging task. 30,[55][56][57][58][59] While successful examples have been reported, the N-terminal cysteine can readily react during gene expression (e.g. pyruvate) limiting the potential FPBA labeling reaction.…”

“…pyruvate) limiting the potential FPBA labeling reaction. 30,[55][56][57][58][59] Instead, this cheap and commercially available reagent can be used as a scavenger for AEP catalysis. We propose that the nonnatural secondary amine motif formed between FPBA and Nterminal cysteine is unlikely to be a reactive substrate for AEP catalysis, and thus the byproduct of the enzymatic reaction Cys-Leu can be potentially trapped by the addition of FPBA.…”

Host defence peptides identified in human apolipoprotein B as promising antifungal agents

“…Expression and isolation of recombinant peptides were performed as described previously (Gaglione et al 2017;Pane et al 2018b;Gaglione et al 2019b;Gaglione et al 2020). ApoB-derived peptides' sequences are reported in Table 1.…”

“…Pro was obtained by chemical hydrolysis of purified ONC-DCless-H6-(C)-ApoB L Pro fusion protein in 5 M gua nidine-HCl co ntaining 1 m M T CEP (tris(2carboxyethyl)phosphine) at pH 7.4. A chimeric construct was expressed and purified as previously reported (Gaglione et al 2017;Pane et al 2018b;Gaglione et al 2019b;Gaglione et al 2020). Peptide release was monitored by reversed-phase high-performance liquid chromatography (RP-HPLC) carried out by using a Jasco LC-4000 system equipped with PU-4086 semipreparative pumps and MD-4010 photo diode array detector.…”