Production and cleavage of a fusion protein of porcine trypsinogen and enhanced green fluorescent protein (EGFP) in Pichia pastoris

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F60461373%3A22330%2F18%3A43917114" target="_blank" >RIV/60461373:22330/18:43917114 - isvavai.cz</a>

Výsledek na webu

<a href="https://link.springer.com/article/10.1007%2Fs12223-018-0619-y" target="_blank" >https://link.springer.com/article/10.1007%2Fs12223-018-0619-y</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1007/s12223-018-0619-y" target="_blank" >10.1007/s12223-018-0619-y</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Production and cleavage of a fusion protein of porcine trypsinogen and enhanced green fluorescent protein (EGFP) in Pichia pastoris

Popis výsledku v původním jazyce

Pharmaceutical grade trypsin is in ever-increasing demand for medical and industrial applications. Improving the efficiency of existing biotechnological manufacturing processes is therefore paramount. When produced biotechnologically, trypsinogenthe inactive precursor of trypsinis advantageous, since active trypsin would impair cell viability. To study factors affecting cell physiology and the production of trypsinogen in fed-batch cultures, we built a fusion protein of porcine trypsinogen and enhanced green fluorescent protein (EGFP) in Pichia pastoris. The experiments were performed with two different pH values (5.0 and 5.9) and two constant specific growth rates (0.02 and 0.04 1/h), maintained using exponential addition of methanol. All the productivity data presented rely on an active determination of trypsin obtained by proteolysis of the trypsinogen produced. The pH of the medium did not affect cell growth, but significantly influenced specific production of trypsinogen: A 1.7-fold higher concentration of trypsinogen was achieved at pH5.9 (64mg/L at 0.02 1/h) compared to pH5.0. EGFP was primarily used to facilitate detection of intracellular protein over the biosynthetic time course. Using flow cytometry with fluorescence detection, cell disruption was avoided, and protein extraction and purification prior to analysis were unnecessary. However, Western blot and SDS-PAGE showed that cleavage of EGFP-trypsinogen fusion protein occurred, probably caused by Pichia-endogenous proteases. The fluorescence analysis did therefore not accurately represent the actual trypsinogen concentration. However, we gained new experimentally-relevant insights, which can be used to avoid misinterpretation of tracking and quantifying as well as online-monitoring of proteins with the frequently used fluorescent tags.

Název v anglickém jazyce

Production and cleavage of a fusion protein of porcine trypsinogen and enhanced green fluorescent protein (EGFP) in Pichia pastoris

Popis výsledku anglicky

Pharmaceutical grade trypsin is in ever-increasing demand for medical and industrial applications. Improving the efficiency of existing biotechnological manufacturing processes is therefore paramount. When produced biotechnologically, trypsinogenthe inactive precursor of trypsinis advantageous, since active trypsin would impair cell viability. To study factors affecting cell physiology and the production of trypsinogen in fed-batch cultures, we built a fusion protein of porcine trypsinogen and enhanced green fluorescent protein (EGFP) in Pichia pastoris. The experiments were performed with two different pH values (5.0 and 5.9) and two constant specific growth rates (0.02 and 0.04 1/h), maintained using exponential addition of methanol. All the productivity data presented rely on an active determination of trypsin obtained by proteolysis of the trypsinogen produced. The pH of the medium did not affect cell growth, but significantly influenced specific production of trypsinogen: A 1.7-fold higher concentration of trypsinogen was achieved at pH5.9 (64mg/L at 0.02 1/h) compared to pH5.0. EGFP was primarily used to facilitate detection of intracellular protein over the biosynthetic time course. Using flow cytometry with fluorescence detection, cell disruption was avoided, and protein extraction and purification prior to analysis were unnecessary. However, Western blot and SDS-PAGE showed that cleavage of EGFP-trypsinogen fusion protein occurred, probably caused by Pichia-endogenous proteases. The fluorescence analysis did therefore not accurately represent the actual trypsinogen concentration. However, we gained new experimentally-relevant insights, which can be used to avoid misinterpretation of tracking and quantifying as well as online-monitoring of proteins with the frequently used fluorescent tags.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

20902 - Bioprocessing technologies (industrial processes relying on biological agents to drive the process) biocatalysis, fermentation

Projekt

—

Návaznosti

R - Projekt Ramcoveho programu EK

Rok uplatnění

2018

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Folia Microbiologica

ISSN

0015-5632

e-ISSN

—

Svazek periodika

63

Číslo periodika v rámci svazku

6

Stát vydavatele periodika

NL - Nizozemsko

Počet stran výsledku

15

Strana od-do

773-787

Kód UT WoS článku

000445889300014

EID výsledku v databázi Scopus

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