Document Detail

Engineering HlyA hypersecretion in Escherichia coli based on proteomic and microarray analyses.
MedLine Citation:
PMID:  15580578     Owner:  NLM     Status:  MEDLINE    
Escherichia coli is a common host for recombinant protein production for biotechnology applications. Secretion to the extracellular media has the potential to reduce protein aggregation and to simplify downstream purification. However, the complexity of the mechanisms of protein secretion has confounded prior attempts to engineer enhanced secretion phenotypes. Here, mutagenesis was used to perturb E. coli W3110 cells secreting HlyA via a Type I pathway. An activity assay identified a mutant secreting fourfold more active alpha-hemolysin than the parent strain. The mutant was characterized using both high-density microarrays for mRNA profiling and a proteomics strategy for protein expression. The relative mRNA and protein expression levels of tRNA-synthetases were decreased in the mutant compared to the parent. A mathematical model of prokaryotic translation was used to design a variant of the hlyA gene that encodes the same amino acid sequence but uses rare codons to slow the rate of translation by altering five bases. Analysis of the parent strain transformed with a plasmid containing this variant gene resulted in the recovery of, and further improvement upon, the selected hypersecretion phenotype. These results present one of the first successful metabolic engineering attempts based on molecular information provided by mRNA and protein expression profiling approaches and resulting in a phenotype useful to the biotechnology community.
Pat S Lee; Kelvin H Lee
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, Non-P.H.S.    
Journal Detail:
Title:  Biotechnology and bioengineering     Volume:  89     ISSN:  0006-3592     ISO Abbreviation:  Biotechnol. Bioeng.     Publication Date:  2005 Jan 
Date Detail:
Created Date:  2004-12-27     Completed Date:  2005-06-30     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  7502021     Medline TA:  Biotechnol Bioeng     Country:  United States    
Other Details:
Languages:  eng     Pagination:  195-205     Citation Subset:  IM    
School of Chemical and Biomolecular Engineering, Cornell University, 120 Olin Hall, Ithaca, New York 14853-5201, USA.
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MeSH Terms
Computer Simulation
Escherichia coli / genetics*,  metabolism*
Escherichia coli Proteins / biosynthesis*,  genetics*
Gene Expression Profiling / methods*
Gene Expression Regulation, Bacterial / genetics
Genetic Enhancement / methods
Hemolysin Proteins / biosynthesis*,  genetics*
Models, Genetic
Mutagenesis, Site-Directed / genetics
Oligonucleotide Array Sequence Analysis / methods*
Protein Engineering / methods*
Proteomics / methods
Recombinant Proteins / biosynthesis
Reg. No./Substance:
0/Escherichia coli Proteins; 0/Hemolysin Proteins; 0/Hlya protein, E coli; 0/Recombinant Proteins

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

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