Determination of an Optimal Temperature Range of Methionine Aminopeptidase from Thermus thermophilus
https://doi.org/10.33647/2713-0428-20-3E-20-24
Abstract
Removal of the N-terminal initiator methionine (Met1) affects 50–70% of cellular proteins, being an important post-translational modification. This modification is catalyzed by such specific enzymes as methionine aminopeptidases (MAP). Recombinant MAPs from E. coli and humans are used in scientific research, as well as in biotechnology to remove Met1 from recombinant proteins under their heterologous overexpression in E. coli. Differences in substrate specificity and operating conditions of known MAPs warrants the search for new enzymes capable of operating at elevated temperatures. We cloned an MAP from a hyperthermophilic bacterium, developed a method for purifying the enzyme, and studied the activity of the enzyme in a wide temperature range. We show that the MAP from Thermus thermophilus is most active at temperatures from 75 to 95°С. The new enzyme can be used to remove N-terminal methionine from recombinant proteins in vitro at elevated temperatures.
Keywords
methionine aminopeptidase,
Thermus thermophilus,
heterologous expression,
N-terminal methionine excision
Supporting agencies: research was funded by the Russian Science Foundation, grant No. 23-24-00563.
About the Authors
V. V. Bykov
Institute of Biological Instrumentation, Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences
Russian Federation
Russian Federation
Vyacheslav V. Bykov
142290, Moscow Region, Pushchino, Nauki Ave., 3
A. A. Vologzhannikova
Institute of Biological Instrumentation, Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences
Russian Federation
Russian Federation
Alisa A. Vologzhannikova, Cand. Sci. (Biol.)
142290, Moscow Region, Pushchino, Nauki Ave., 3
M. V. Trunilina
Institute of Biological Instrumentation, Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences
Russian Federation
Russian Federation
Maria V. Trunilina
142290, Moscow Region, Pushchino, Nauki Ave., 3
T. A. Kudryashov
Institute of Biological Instrumentation, Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences
Russian Federation
Russian Federation
Timofey A. Kudryashov
142290, Moscow Region, Pushchino, Nauki Ave., 3
Yu. S. Lapteva
Institute of Biological Instrumentation, Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences
Russian Federation
Russian Federation
Yulia S. Lapteva, Cand. Sci. (Biol.)
142290, Moscow Region, Pushchino, Nauki Ave., 3
A. S. Sokolov
Institute of Biological Instrumentation, Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences
Russian Federation
Russian Federation
Andrey S. Sokolov, Cand. Sci. (Biol.)
142290, Moscow Region, Pushchino, Nauki Ave., 3
References
1. Lapteva Yu.S., Bykov V.V., Trunilina M.V., Boldaevsky I.S., Kudryashov T.A., Vologzhannikova A.A., Sokolov A.S. Poluchenie sverkhstabil'noy metioninaminopeptidazy dlya udale-niya metionina iz rekombinantnykh belkov [Obtaining overstable methionine aminopeptidase for the removal of methionine from recombinant proteins]. Biomeditsina [Journal Biomed]. 2023;19(3Е):47–51. (In Russian). DOI: 10.33647/2713-0428-19-3Е-47-51
2. Adachi K., Yamaguchi T., Yang Y., Konitzer P.T., Pang J., Reddy K.S., Ivanova M., Ferrone F., Surrey S. Expression of functional soluble human alpha-globin chains of hemoglobin in bacteria. Protein Exp. Purif. 2000;20(1):37–44. DOI: 10.1006/prep.2000.1277
3. Boix E., Wu Y., Vasandani V.M., Saxena S.K., Ardelt W., Ladner J., Youle R.J. Role of the N terminus in RNase A homologues: Differences in catalytic activity, ribonuclease inhibitor interaction and cytotoxicity. J. Mol. Biol. 1996;257(5):992–1007. DOI: 10.1006/jmbi.1996.0218
4. Endo S., Yamamoto Y., Sugawara T., Nishimura O., Fujino M. The additional methionine residue at the N-terminus of bacterially expressed human interleukin-2 affects the interaction between the N- and C-termini. Biochemistry. 2001;40(4):914–919. DOI: 10.1021/bi001170r
5. Giglione C., Boularot A., Meinnel T. Protein N-terminal methionine excision. Cell Mol. Life Sci. 2004;61(12):1455–1474. DOI: 10.1007/s00018-004-3466-8
6. Helgren T.R., Chen C., Wangtrakuldee P., EdwardsT.E., Staker B.L., Abendroth J., Sankaran B., Housley N.A., Myler P.J., Audia J.P., Horn J.R. Hagen T.J. Rickettsia prowazekii methionine aminopeptidase as a promising target for the development of antibacterial agents. Bioorg. Med. Chem. 2017;25(3):813–824. DOI: 10.1016/j.bmc.2016.11.013
7. Liao Y.D., Wang S.C., Leu Y.J., Wang C.F., Chang S.T., Hong Y.T., Pan Y.R., Chen C. The structural integrity exerted by N-terminal pyroglutamate is crucial for the cytotoxicity of frog ribonuclease from Rana pipiens. Nucleic Acids Res. 2003;31(18):5247–5255. DOI: 10.1093/nar/gkg746
8. Selvakumar P., Lakshmikuttyamma A., Dimmock J.R., Sharma R.K. Methionine aminopeptidase 2 and cancer. Biochim. Biophys. Acta. 2006;
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For citations:
Bykov V.V., Vologzhannikova A.A., Trunilina M.V., Kudryashov T.A., Lapteva Yu.S., Sokolov A.S. Determination of an Optimal Temperature Range of Methionine Aminopeptidase from Thermus thermophilus. Journal Biomed. 2024;20(3E):20-24. (In Russ.) https://doi.org/10.33647/2713-0428-20-3E-20-24
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