Document Detail


Use of the green fluorescent protein variant (YFP) to monitor MetArg human proinsulin production in Escherichia coli.
MedLine Citation:
PMID:  10419827     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Green fluorescent protein (GFP), a relatively new reporter gene, is making an impact on many aspects of science. The attributes of GFP could also be applied to the area of recombinant protein production. The work described here represents the first experiments using GFP as a tool to monitor recombinant protein production in real time in the fermentation process. We have constructed plasmids containing an operon fusion of the gene encoding MetArg-human proinsulin and reporter gene GFP (GFP, BFP, and YFP variants). The MetArg-proinsulin and GFP variant reporter protein were overexpressed in Escherichia coli BL21(DE3) after isopropyl beta-d-thiogalactoside induction. The MetArg-proinsulin to YFP protein ratio did not change in the cells during the bioprocess. Since there is a quantitative relationship between the level of MetArg-proinsulin concentration and YFP fluorescence, it is possible to measure only YFP fluorescence in order to monitor the production of MetArg-proinsulin during the bioprocess. The expression level of MetArg-proinsulin could reach 20-25%. Some 140 mg recombinant MetArg-human proinsulin could be obtained easily from 1 liter of fermentation medium. The MetArg-proinsulin could simply be changed into human insulin by trypsin and carboxypeptidase B treatment in later steps. These experiments provide possibilities for using the YFP reporter gene as a convenient tool to monitor protein expression in biotechnological processes. The proposed technique could reduce the time- and labor-intensive analysis of protein production and would improve the efficiency of process development.
Authors:
S Daabrowski; A Brillowska; J Kur
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Protein expression and purification     Volume:  16     ISSN:  1046-5928     ISO Abbreviation:  Protein Expr. Purif.     Publication Date:  1999 Jul 
Date Detail:
Created Date:  1999-08-18     Completed Date:  1999-08-18     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  9101496     Medline TA:  Protein Expr Purif     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  315-23     Citation Subset:  IM    
Copyright Information:
Copyright 1999 Academic Press.
Affiliation:
Department of Microbiology, Technical University of Gdańsk, ul. Narutowicza 11/12, Gdańsk, 80-952, Poland.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Arginine / chemistry*
Base Sequence
Cloning, Molecular
DNA, Recombinant
Escherichia coli / genetics
Green Fluorescent Proteins
Humans
Luminescent Proteins / genetics*
Methionine / chemistry*
Molecular Sequence Data
Plasmids
Proinsulin / chemistry,  genetics*
Chemical
Reg. No./Substance:
0/DNA, Recombinant; 0/Luminescent Proteins; 147336-22-9/Green Fluorescent Proteins; 63-68-3/Methionine; 74-79-3/Arginine; 9035-68-1/Proinsulin

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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