Gene Analysis, Cloning, and Heterologous Expression of Protease from a Micromycete Aspergillus ochraceus Capable of Activating Protein C of Blood Plasma
Abstract
:1. Introduction
2. Materials and Methods
2.1. Microorganism and Growth Conditions
2.2. cDNA Sequencing and Cloning of PAPC-4104
2.3. In Silico Protein Analysis
2.4. Alignments and Phylogeny
2.5. Construction of the Expression Plasmid
2.6. Expression and Purification of Recombinant PAPC-4104
2.7. Purification of Native PAPC-4104
2.8. Enzyme Activity Assays
2.9. General Analytical Methods
3. Results
3.1. Gene Organization and Protein Sequence
3.2. Tertiary Structure and Active Site Residue
3.3. Phylogeny and Evolution
3.4. Properties of the Recombinant PAPC-4104
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Substrate | Activity of Recombinant PAPC-4104 | Activity of Native PAPC-4104 |
---|---|---|
Conjugate reactions (with blood plasma) | ||
pGlu-Pro-Arg-pNA | + | + |
Z-D-Arg-Gly-Arg-pNA | + | + |
Direct reactions (without blood plasma) | ||
pGlu-Pro-Arg-pNA | − | − |
Z-D-Arg-Gly-Arg-pNA | − | − |
H-D-Val-Leu-Lys-pNA | + | + |
Tos-Gly-Pro-Arg-pNA | + | + |
H-D-Pro-Phe-Arg-pNA | − | − |
H-D-Phe-Pip-Arg-pNA | − | − |
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Komarevtsev, S.K.; Evseev, P.V.; Shneider, M.M.; Popova, E.A.; Tupikin, A.E.; Stepanenko, V.N.; Kabilov, M.R.; Shabunin, S.V.; Osmolovskiy, A.A.; Miroshnikov, K.A. Gene Analysis, Cloning, and Heterologous Expression of Protease from a Micromycete Aspergillus ochraceus Capable of Activating Protein C of Blood Plasma. Microorganisms 2021, 9, 1936. https://doi.org/10.3390/microorganisms9091936
Komarevtsev SK, Evseev PV, Shneider MM, Popova EA, Tupikin AE, Stepanenko VN, Kabilov MR, Shabunin SV, Osmolovskiy AA, Miroshnikov KA. Gene Analysis, Cloning, and Heterologous Expression of Protease from a Micromycete Aspergillus ochraceus Capable of Activating Protein C of Blood Plasma. Microorganisms. 2021; 9(9):1936. https://doi.org/10.3390/microorganisms9091936
Chicago/Turabian StyleKomarevtsev, Sergei K., Peter V. Evseev, Mikhail M. Shneider, Elizaveta A. Popova, Alexey E. Tupikin, Vasiliy N. Stepanenko, Marsel R. Kabilov, Sergei V. Shabunin, Alexander A. Osmolovskiy, and Konstantin A. Miroshnikov. 2021. "Gene Analysis, Cloning, and Heterologous Expression of Protease from a Micromycete Aspergillus ochraceus Capable of Activating Protein C of Blood Plasma" Microorganisms 9, no. 9: 1936. https://doi.org/10.3390/microorganisms9091936
APA StyleKomarevtsev, S. K., Evseev, P. V., Shneider, M. M., Popova, E. A., Tupikin, A. E., Stepanenko, V. N., Kabilov, M. R., Shabunin, S. V., Osmolovskiy, A. A., & Miroshnikov, K. A. (2021). Gene Analysis, Cloning, and Heterologous Expression of Protease from a Micromycete Aspergillus ochraceus Capable of Activating Protein C of Blood Plasma. Microorganisms, 9(9), 1936. https://doi.org/10.3390/microorganisms9091936