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Preparation and Properties of the Recombinant Tenebrio molitor SerPH122—Proteolytically Active Homolog of Serine Peptidase

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Abstract

Pseudoenzymes are homologs of active enzymes that have amino acid substitutions in the active center and, therefore, usually do not possess enzymatic activity. In this work, a recombinant proprotein (proSerPH122) of the homolog of serine peptidases of the S1 family from the yellow mealworm (Tenebrio molitor) was obtained in the yeast producer strain Komagataella kurtzmanii. The target His6-tagged protein was produced in a glycosylated form during secretion in yeast. The properties of both glycosylated and deglycosylated forms were studied. The proSerPH122 homolog with the replacement of active site Ser with Thr was pretreated with trypsin to study the enzymatic properties. The processed, mature homolog SerPH122 was shown to have low, but reliably detectable activity on the chromogenic substrate Suc-Ala-Ala-Pro-Phe-pNA, and this activity did not depend on the glycosylation level.

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ACKNOWLEDGMENTS

The authors are grateful to M.V. Serebryakova for the mass-spectrometric analysis of the preparation of the recombinant prohomolog proSerPH122.

Funding

The study was carried out with the financial support of a grant from the Russian Science Foundation (project no. 19-74-00080).

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Authors and Affiliations

  1. Lomonosov Moscow State University, Department of Chemistry, 119991, Moscow, Russia

    V. F. Tereshchenkova & I. Yu. Filippova

  2. Lomonosov Moscow State University, Faculty of Biology, 119991, Moscow, Russia

    N. I. Zhiganov & N. V. Belyaeva

  3. National Research Center “Kurchatov Institute”—GOSNIIGENETIKA, Kurchatov Genomic Center, 117545, Moscow, Russia

    P. I. Akentyev & I. I. Gubaidullin

  4. National Research Center “Kurchatov Institute”, 123182, Moscow, Russia

    P. I. Akentyev, I. I. Gubaidullin & D. G. Kozlov

  5. Lomonosov Moscow State University, A.N. Belozersky Research Institute of Physico-Chemical Biology, 119991, Moscow, Russia

    E. N. Elpidina

Authors
  1. V. F. Tereshchenkova
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  2. N. I. Zhiganov
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  3. P. I. Akentyev
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  4. I. I. Gubaidullin
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  5. D. G. Kozlov
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  6. N. V. Belyaeva
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  7. I. Yu. Filippova
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  8. E. N. Elpidina
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Corresponding author

Correspondence to E. N. Elpidina.

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The authors declare that they have no conflict of interest. This article does not contain any studies involving animals or human participants performed by any of the authors.

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Tereshchenkova, V.F., Zhiganov, N.I., Akentyev, P.I. et al. Preparation and Properties of the Recombinant Tenebrio molitor SerPH122—Proteolytically Active Homolog of Serine Peptidase. Appl Biochem Microbiol 57, 579–585 (2021). https://doi.org/10.1134/S0003683821050161

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