Document Detail


Green fluorescent protein variants fold differentially in prokaryotic and eukaryotic cells.
MedLine Citation:
PMID:  11455577     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Better-folding Green Fluorescent Protein (GFP) mutants selected from bacterial screenings are commonly used in widely different cellular environments. However, it is unclear if the folding efficiency of GFPs is invariant in different cell types. In this work, we have analysed the folding properties of GFP variants in bacteria versus mammalian cells. Remarkably, S65T was found to fold at comparable levels with the wild type GFP in bacteria, but at 10-fold lower levels in mammalian cells. On the other hand, Bex1 folded 3-4 times better than the wtGFP or S65T in E. coli, and 10-20-fold or more than 95-fold better, respectively, in mammalian cells. The Vex1 mutant demonstrated similar properties to Bex1. No evidence of differential GFP unfolding in vivo or of preferential degradation of unfolded GFP molecules was found. Moreover, no relationship between GFP folding efficiency and expression levels, or protein stability was detected. Trivial Aconfounding factors, like GFP unfolding caused by different pH or fluorescence quenching due to molecular crowding, were also excluded. In summary, our results demonstrate that specific GFP variants follow different folding trajectories in mammalian versus bacterial cells. The specificity of this differential folding supports a role of chaperones in guiding the folding of GFP in vivo. J. Cell. Biochem. Suppl. 36: 117-128, 2001.
Authors:
A Sacchetti; V Cappetti; P Marra; R Dell'Arciprete; T El Sewedy; C Crescenzi; S Alberti
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Publication Detail:
Type:  Comparative Study; Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Journal of cellular biochemistry. Supplement     Volume:  Suppl 36     ISSN:  0733-1959     ISO Abbreviation:  J. Cell. Biochem. Suppl.     Publication Date:  2001  
Date Detail:
Created Date:  2001-07-16     Completed Date:  2001-09-13     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  8207539     Medline TA:  J Cell Biochem Suppl     Country:  United States    
Other Details:
Languages:  eng     Pagination:  117-28     Citation Subset:  IM    
Copyright Information:
Copyright 2001 Wiley-Liss, Inc.
Affiliation:
Biotech group - Laboratory of Experimental Oncology, Department of Cell Biology and Oncology, Istituto di Ricerche Farmacologiche Mario Negri, 66030 Santa Maria Imbaro (Chieti), Italy.
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MeSH Terms
Descriptor/Qualifier:
Animals
Bacteria / chemistry*,  cytology,  metabolism
Cell Line
Cercopithecus aethiops
Eukaryotic Cells
Flow Cytometry
Green Fluorescent Proteins
Humans
Luminescent Proteins / chemistry*,  genetics,  metabolism
Mice
Microscopy, Fluorescence
Mutation
Protein Folding*
Spectrometry, Fluorescence
Chemical
Reg. No./Substance:
0/Luminescent Proteins; 147336-22-9/Green Fluorescent Proteins

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