Characterization of Nicotiana benthamiana subtilisin-like peptidases

master's thesis

• Title (croatian): Karakterizacija peptidaza sličnih subtilizinu iz biljke Nicotiana benthamiana
• Author: Ana Vrkić
• Mentor: Ksenija Durgo (mentor)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Biochemical Engineering); (Laboratory for Biology and Microbial Genetics); Zagreb
• Defense date and country: 2021-09-30, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Despite its many advantages, production of recombinant proteins in plants often encounters low yields, mainly due to proteolytic degradation by endogenous peptidases. Nicotiana benthamiana, a promising expression platform for production of human biologics is no exception. It was previously found that broadly neutralizing antibodies (bnAbs) against type 1 human immunodeficiency virus (HIV-1) (2F5, PG9) undergo proteolytic degradation when expressed in N. benthamiana leaves, and recently, two subtilisin-like serine peptidases were found to be responsible for the majority of those cleavage events. In this thesis, two Nb subtilases (NbPhyt1 and NbP69F) suspected to be involved in proteolysis of anti-HIV-1 bnAbs in planta, were characterized. It was shown that in vitro NbPhyt1 cleaves the PG9 heavy chain at the third complementarity determining region (CDRH3), at the same position as the Ab is cleaved in planta (NYYD112↓F113YDG). The enzyme is most reactive towards the Ab at pH 4.0-5.5, which is in accordance with the enzyme's activity in the apoplastic fluid (pH~5.5). NbPhyt1 activity towards PG9 was best inhibited by fluorophosphonate-biotin (FP-biotin) and N-acetyl-YVAD chloromethyl ketone (Ac-YVAD-CMK), then 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) and benzyloxycarbonyl-VAD fluoromethyl ketone (Z-VAD-FMK). The collected data strongly suggest that NbPhyt1 participates in processing of PG9 in planta

Abstract (english)

Unatoč mnogim prednostima biljaka u proizvodnji rekombinantnih proteina, njihov najveći nedostatak je nizak prinos proizvoda za koji su uglavnom odgovorne biljne endogene peptidaze. S istim se problemom susreće i Nicotiana benthamiana, obećavajući domaćin za proizvodnju rekombinantnih ljudskih proteina. Prethodno je pokazano da neutralizirajuća antitijela protiv virusa humane imunodeficijencije tipa 1 (HIV-1) (2F5, PG9) podliježu razgradnji prilikom proizvodnje u listovima ove biljke, te je nedavno utvrđeno da su za proteolizu velikim dijelom odgovorne dvije subtilizinu slične serinske peptidaze (subtilaze). U ovom radu su okarakterizirane dvije Nb subtilaze (NbPhyt1 i NbP69F) za koje se sumnja da sudjeluju u proteolizi neutraliziraućih anti-HIV-1 antitijela in planta. Rezultati pokazuju da, in vitro, NbPhyt1 cijepa teški lanac PG9 antitijela u trećoj regiji koja određuje komplementarnost (CDRH3), na istom mjestu kao i kod antitijela proizvedenog in planta (NYYD112↓F113YDG). Najveća aktivnost NbPhyt1 je utvrđena pri pH 4.0-5.5, što je u skladu s aktivnosti enzima u apoplastu (pH~5.5). Najbolje inhibitorno djelovanje na aktivnost NbPhyt1 imali su fluorofosfonat-biotin (FP-biotin) i N-acetil-YVAD klorometil keton (Ac-YVAD-CMK), zatim 4-(2-aminoetil)benzensulfonil fluorid hidroklorid (AEBSF) i benziloksikarbonil-VAD fluorometil keton (Z-VAD-FMK). Prikupljeni rezultati snažno upućuju na to da NbPhyt1 sudjeluje u razgradnji PG9 antitijela in planta.

• Keywords: Nicotiana benthamiana; monoclonal antibody; peptidase
• Keywords (english): Nicotiana benthamiana; monoklonalno antitijelo; peptidaza
• Language: english
• URN:NBN: urn:nbn:hr:159:510496
• Study programme: Title: Molecular Biotechnology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka molekularne biotehnologije (magistar/magistra inženjer/inženjerka molekularne biotehnologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2022-05-26 12:44:09