Production of bioactive peptides in an in vitro system
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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