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

Result code in 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>

Result on the web

<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>

Result language

angličtina

Original language name

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

Original language description

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.

Czech name

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Czech description

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Type

J_imp - Article in a specialist periodical, which is included in the Web of Science database

CEP classification

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OECD FORD branch

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

Project

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Continuities

R - Projekt Ramcoveho programu EK

Publication year

2018

Confidentiality

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

Name of the periodical

Folia Microbiologica

ISSN

0015-5632

e-ISSN

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Volume of the periodical

63

Issue of the periodical within the volume

6

Country of publishing house

NL - THE KINGDOM OF THE NETHERLANDS

Number of pages

15

Pages from-to

773-787

UT code for WoS article

000445889300014

EID of the result in the Scopus database

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