Expression of acid cleavable Asp-Pro linked multimeric AFP peptide in E. coli.
Murad Mollaev, Artur Zabolotskii, Neonila Gorokhovets, Elena Nikolskaya, Maria Sokol, Andrey Tsedilin, Mariia Mollaeva, Margarita Chirkina, Timofey Kuvaev, Anna Pshenichnikova, Nikita Yabbarov
Author Information
Murad Mollaev: Biotechnology and Industrial Pharmacy Department, Lomonosov Institute of Fine Chemical Technologies, MIREA - Russian Technological University, 86 Vernadsky avenue, Moscow, 119454, Russia.
Artur Zabolotskii: JSC Russian Research Center for Molecular Diagnostics and Therapy, 8 Simferopolsky boulevard, Moscow, 117638, Russia.
Neonila Gorokhovets: I.M. Sechenov First Moscow State Medical University, 8-2 Trubetskaya street, Moscow, 119991, Russia.
Elena Nikolskaya: JSC Russian Research Center for Molecular Diagnostics and Therapy, 8 Simferopolsky boulevard, Moscow, 117638, Russia.
Maria Sokol: JSC Russian Research Center for Molecular Diagnostics and Therapy, 8 Simferopolsky boulevard, Moscow, 117638, Russia.
Andrey Tsedilin: Fundamentals of Biotechnology Federal Research Center, RAS, 33 Leninsky avenue, Moscow, 119071, Russia.
Mariia Mollaeva: JSC Russian Research Center for Molecular Diagnostics and Therapy, 8 Simferopolsky boulevard, Moscow, 117638, Russia.
Margarita Chirkina: JSC Russian Research Center for Molecular Diagnostics and Therapy, 8 Simferopolsky boulevard, Moscow, 117638, Russia.
Timofey Kuvaev: National Research Center "Kurchatov Institute", Research Institute for Genetics and Selection of Industrial Microorganisms, 1 1-Y Dorozhnyy Proyezd, Moscow, 117545, Russia.
Anna Pshenichnikova: Biotechnology and Industrial Pharmacy Department, Lomonosov Institute of Fine Chemical Technologies, MIREA - Russian Technological University, 86 Vernadsky avenue, Moscow, 119454, Russia.
Nikita Yabbarov: JSC Russian Research Center for Molecular Diagnostics and Therapy, 8 Simferopolsky boulevard, Moscow, 117638, Russia. marvint@inbox.ru. ORCID
BACKGROUND: Difficult to express peptides are usually produced by co-expression with fusion partners. In this case, a significant mass part of the recombinant product falls on the subsequently removed fusion partner. On the other hand, multimerization of peptides is known to improve its proteolytic stability in E. coli due to the inclusion of body formation, which is sequence specific. Thereby, the peptide itself may serve as a fusion partner and one may produce more than one mole of the desired product per mole of fusion protein. This paper proposes a method for multimeric production of a human alpha-fetoprotein fragment with optimized multimer design and processing. This fragment may further find its application in the cytotoxic drug delivery field or as an inhibitor of endogenous alpha-fetoprotein. RESULTS: Multimerization of the extended alpha-fetoprotein receptor-binding peptide improved its stability in E. coli, and pentamer was found to be the largest stable with the highest expression level. As high as 10 aspartate-proline bonds used to separate peptide repeats were easily hydrolyzed in optimized formic acid-based conditions with 100% multimer conversion. The major product was represented by unaltered functional alpha-fetoprotein fragment while most side-products were its formyl-Pro, formyl-Tyr, and formyl-Lys derivatives. Single-step semi-preparative RP-HPLC was enough to separate unaltered peptide from the hydrolysis mixture. CONCLUSIONS: A recombinant peptide derived from human alpha-fetoprotein can be produced via multimerization with subsequent formic acid hydrolysis and RP-HPLC purification. The reported procedure is characterized by the lower reagent cost in comparison with enzymatic hydrolysis of peptide fusions and solid-phase synthesis. This method may be adopted for different peptide expression, especially with low amino and hydroxy side chain content.
