Elsevier

Analytical Biochemistry

Volume 366, Issue 2, 15 July 2007, Pages 182-189
Analytical Biochemistry

Production of bioactive peptides in an in vitro system

https://doi.org/10.1016/j.ab.2007.04.020Get rights and content

Abstract

An in vitro system for the preparation of bioactive peptides is described. This system couples three different posttranslational modification enzymes, prohormone convertases (PCs), carboxypeptidase E, and peptidyl α-amidating enzyme, to transform recombinant precursors into bioactive peptides. Three different precursors, mouse proopiomelanocortin (mPOMC), rat proenkephalin (rPE), and human proghrelin, were used as model systems. The conversion of mPOMC and rPE to smaller peptide products was measured by radioimmunoassay. After optimization of the system, excellent efficiency was obtained: about 85% of starting mPOMC was converted to des-acetyl α-melanocyte-stimulating hormone (α-MSH). For proenkephalin, 75 and 96% yields were obtained for the opioid peptides Met-RGL and Met-enk, respectively. Cell-based assays demonstrated that in-vitro-generated des-acetyl α-MSH successfully activated the melanocortin 4 receptor. Proghrelin digestion was used to screen the specificity of PC cleavage and to confirm the cleavage site by mass spectroscopy. Mature ghrelin was produced by human furin, mouse prohormone convertase 1, and human prohormone convertase 7 but not by mouse prohormone convertase 2. These results demonstrate that our in vitro system (1) can produce peptides in quantities sufficient to carry out functional analyses, (2) can be used to determine the specificity of proprotein convertases on recombinant precursors, and (3) has the potential to identify novel peptide functions on both known and orphan G-protein-coupled receptors.

Section snippets

Preparation of recombinant precursors

The preparation of rat proenkephalin (rPE) from Chinese hamster ovary (CHO) cell-conditioned medium has been described previously [23]. For overexpression of mouse POMC (mPOMC) and human proghrelin in Escherichia coli, the coding region for each gene (lacking the signal peptide) was inserted into the pQE-30 vector (Qiagen, Chatsworth, CA) and pProEX (Invitrogen, Carlsbad, CA), respectively. Recombinant precursors were synthesized as His-tagged proteins and then purified on Ni-resin (Sigma,

Preparation of bioactive peptides from rPE and mPOMC

Since PC2 activity was inhibited by both cobalt and ascorbate, which are essential ingredients in the CPE and PAM reactions, respectively [31], [32], we could not perform simultaneous reactions, i.e., endoproteolytic, exoproteolytic, and amidation reactions in a single tube. Therefore, each modification step was first optimized as shown in Fig. 1. Bioactive peptides from mPOMC and rPE were produced by sequential reactions, as shown in Fig. 1. Fig. 2 depicts the efficiency of the production of

Discussion

The data presented here demonstrate that we have successfully processed specific prohormone precursors to mature peptides using an in vitro system. Since ligands for receptors are normally active in the nanomolar range, the amounts of product generated by the above reaction should be sufficient to carry out further studies to test ligand and receptor matching. For example, the quantity of peptides produced by our method (4 ml of 170 nM des-acetyl α-MSH) can activate 136 wells of

Acknowledgments

This work was supported by DA 05084. We thank Rachel Sanders for the construction of the pQE30-POMC vector and Dr. Cristina Caescu for construction of the pPro-proghrelin vector. We are grateful to Dr. Roger Cone for MC4-expressing cells, to Dr. Betty Eipper and Unigene for PAM, and to R&D Systems for PAM and CPE. We thank Dr. David Worthylake for general advice on pPro-Ex vectors and TEV protease cleavage.

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