Large-scale production of functional human adrenomedullin: expression, cleavage, amidation, and purification.
Human adrenomedullin (hAM) is a 52-amino-acid regulatory peptide containing a six-membered ring structure and an amidated C-terminus, features that are essential for its biological activity. Here, we describe a simple and effective protocol for producing large quantities of highly pure, functional recombinant hAM. A peptide precursor (hAM-Gly) was expressed in Escherichia coli as a fusion protein with thioredoxin and collected as inclusion bodies. The fusion protein was then digested with BLase, a glutamate-specific endopeptidase, to prepare hAM-Gly. The essential ring structure formed spontaneously, while the terminal amide was generated by conversion of the added glycine residue using peptidylglycine alpha-amidating enzyme. The low solubility of hAM-Gly enabled the use of a selective precipitation/extraction method to generate a product that was 80-90% pure, which was sufficient to proceed with the alpha-amidating enzyme reaction. The resultant hAM was then purified further by column chromatography. The final yield was about 82 mg/L of bacterial culture, and the purity, determined by reverse phase HPLC, was >99.5%. The recombinant hAM was biologically active, eliciting concentration-dependent increases in cAMP in CHO-K1 cells expressing a specific hAM receptor and hypotensive responses when intravenously injected into rats. This new approach to the synthesis of hAM is simpler and more cost-effective for large-scale production than chemical synthesis. It therefore represents a new powerful tool that has the potential to facilitate analysis of the structure and function of hAM, as well as the development of new therapeutic protocols for the treatment of ailments such as hypertension.
Mitsuda
Yuuichi
Y
Discovery Research Laboratories, Shionogi and Co. Ltd., 2-5-1 Mishima, Settu, Osaka 566-0022, Japan. yuuichi.mitsuda@shionogi.co.jp
Takimoto
Akio
A
Kamitani
Shigeki
S
Kitamura
Kazuo
K
Sakata
Tsuneaki
T
Mitsushima
Kenji
K
D053607:Adrenomedullin; D000577:Amides / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013997:Time Factors
D000577:Amides; D010455:Peptides; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D053607:Adrenomedullin