PRODUCTION OF RECOMBINANT, NON-TAGGED PHENYLALANINE AMMONIA-LYASES EMPLOYING TEV PROTEASE-REMOVABLE AFFINITY TAGS

Alina FILIP; Zsófia BATA; Anca Elena ANGHEL; László POPPE; László Csaba BENCZE

doi:10.24193/subbchem.2022.4.03

PRODUCTION OF RECOMBINANT, NON-TAGGED PHENYLALANINE AMMONIA-LYASES EMPLOYING TEV PROTEASE-REMOVABLE AFFINITY TAGS

Authors

Alina FILIP Enzymology and Applied Biocatalysis Research Center, Faculty of Chemistry and Chemical Engineering, Babeş-Bolyai University of Cluj-Napoca, Arany János str. 11, 400028, Cluj-Napoca, Romania. alina.filip@ubbcluj.ro https://orcid.org/0000-0001-6820-9204
Zsófia BATA Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, Műegyetem rkp. 3, 1111, Budapest, Hungary Institute of Enzymology, ELKH Research Center of Natural Sciences, Magyar tudósok krt. 2, 1117, Budapest, Hungary https://orcid.org/0000-0002-4840-9433
Anca Elena ANGHEL Enzymology and Applied Biocatalysis Research Center, Faculty of Chemistry and Chemical Engineering, Babeş-Bolyai University of Cluj-Napoca, Arany János str. 11, 400028, Cluj-Napoca, Romania
László POPPE Enzymology and Applied Biocatalysis Research Center, Faculty of Chemistry and Chemical Engineering, Babeş-Bolyai University of Cluj-Napoca, Arany János str. 11, 400028, Cluj-Napoca, Romania Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, Műegyetem rkp. 3, 1111, Budapest, Hungary https://orcid.org/0000-0002-8358-1378
László Csaba BENCZE Enzymology and Applied Biocatalysis Research Center, Faculty of Chemistry and Chemical Engineering, Babeş-Bolyai University of Cluj-Napoca, Arany János str. 11, 400028, Cluj-Napoca, Romania. laszlo.bencze@ubbcluj.ro https://orcid.org/0000-0003-0956-9749

DOI:

https://doi.org/10.24193/subbchem.2022.4.03

Keywords:

affinity tags, phenylalanine ammonia-lyase, TEV (Tobacco Etch Virus) protease, thermostability, specific enzyme activity, oligomerization state.

Abstract

Nowadays, protein purification by the aid of affinity tags can be carried out with high speed and efficiency. However, in several cases, affinity tags can significantly alter the key properties of enzymes, especially activity and/or thermostability.

This study focused on the purification of the non-tagged phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL), as well as on the purification of the TEV (Tobacco Etch Virus) protease, the molecular scissors used to remove the affinity tag from the recombinantly expressed PcPAL. Removal of the 6xHis-tag led to a 1.5-fold increase in the specific activity of PcPAL, while the absence of the affinity tag did not significantly alter the thermostability of the protein. The purity and oligomerization state of the proteins of interest were also analyzed by size exclusion chromatography, both before and after the removal of the affinity tag, confirming the stability of the tetrameric fold of PcPAL.

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STUDIA UBB CHEMIA, Volume 67 (LXVII), No. 4, December 2022

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Published

2022-12-30

How to Cite

FILIP, A., BATA, Z., ANGHEL, A. E., POPPE, L., & BENCZE, L. C. (2022). PRODUCTION OF RECOMBINANT, NON-TAGGED PHENYLALANINE AMMONIA-LYASES EMPLOYING TEV PROTEASE-REMOVABLE AFFINITY TAGS. Studia Universitatis Babeș-Bolyai Chemia, 67(4), 27–46. https://doi.org/10.24193/subbchem.2022.4.03