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<span style = 'font-size: large'> '''Engel, CK, Chen, L and Privé, GG'''  (2002) Stability of the lactose permease in detergent solutions.''Biochim. Biophys. Acta'' '''1564''':47-56</span>
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!align=left  |Citation
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'''Engel, CK, Chen, L and Privé, GG'''  (2002) Stability of the lactose permease in detergent solutions. ''Biochim. Biophys. Acta'' '''1564''':47-56
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!align=left  |Abstract
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Protein stability, as measured by irreversible protein aggregation, is one of the central difficulties in the handling of detergent-solubilized membrane proteins. We present a quantitative analysis of the stability of the Escherichia coli lactose (lac) permease and a series of lac permease fusion proteins containing an insertion of cytochrome(b562), T4 lysozyme or beta-lactamase in the central hydrophilic loop of the permease. The stability of the proteins was evaluated under a variety of storage conditions by both a qualitative SDS-PAGE assay and by a quantitative hplc assay. Long-chain maltoside detergents were more effective at maintaining purified protein in solution than detergents with smaller head groups and/or shorter alkyl tails. A full factorial experiment established that the proteins were insensitive to sodium chloride concentrations, but greatly stabilized by glycerol, low temperature and the combination of glycerol and low temperature. The accurate quantitation of the protein by absorbance spectroscopy required exclusion of all contact with clarified polypropylene or polyvinyl chloride (PVC) materials. Although some of the fusion proteins were more prone to aggregation than the wild-type permease, the stability of a fusion protein containing a cytochrome(b562) insertion was indistinguishable from that of native lac permease.
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[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=12100995 PubMed]


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|{{TableHeadingColor}} |'''Abstract''' || Protein stability, as measured by irreversible protein aggregation, is one of the central difficulties in the handling of detergent-solubilized membrane proteins. We present a quantitative analysis of the stability of the Escherichia coli lactose (lac) permease and a series of lac permease fusion proteins containing an insertion of cytochrome(b562), T4 lysozyme or beta-lactamase in the central hydrophilic loop of the permease. The stability of the proteins was evaluated under a variety of storage conditions by both a qualitative SDS-PAGE assay and by a quantitative hplc assay. Long-chain maltoside detergents were more effective at maintaining purified protein in solution than detergents with smaller head groups and/or shorter alkyl tails. A full factorial experiment established that the proteins were insensitive to sodium chloride concentrations, but greatly stabilized by glycerol, low temperature and the combination of glycerol and low temperature. The accurate quantitation of the protein by absorbance spectroscopy required exclusion of all contact with clarified polypropylene or polyvinyl chloride (PVC) materials. Although some of the fusion proteins were more prone to aggregation than the wild-type permease, the stability of a fusion protein containing a cytochrome(b562) insertion was indistinguishable from that of native lac permease.
!align=left  |Keywords
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Chromatography, High Pressure Liquid; Cytochrome b Group/chemistry; Detergents; Drug Stability; Electrophoresis, Polyacrylamide Gel; Escherichia coli/chemistry; Escherichia coli/genetics; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Glycerol; Membrane Transport Proteins/chemistry; Membrane Transport Proteins/genetics; Monosaccharide Transport Proteins; Recombinant Fusion Proteins/chemistry; Recombinant Fusion Proteins/genetics; Solutions; Spectrophotometry, Ultraviolet; Symporters; Temperature
|{{TableHeadingColor}} nowrap |'''Links''' ||  [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=12100995 PubMed]
 
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Latest revision as of 04:17, 15 September 2013

Citation

Engel, CK, Chen, L and Privé, GG (2002) Stability of the lactose permease in detergent solutions. Biochim. Biophys. Acta 1564:47-56

Abstract

Protein stability, as measured by irreversible protein aggregation, is one of the central difficulties in the handling of detergent-solubilized membrane proteins. We present a quantitative analysis of the stability of the Escherichia coli lactose (lac) permease and a series of lac permease fusion proteins containing an insertion of cytochrome(b562), T4 lysozyme or beta-lactamase in the central hydrophilic loop of the permease. The stability of the proteins was evaluated under a variety of storage conditions by both a qualitative SDS-PAGE assay and by a quantitative hplc assay. Long-chain maltoside detergents were more effective at maintaining purified protein in solution than detergents with smaller head groups and/or shorter alkyl tails. A full factorial experiment established that the proteins were insensitive to sodium chloride concentrations, but greatly stabilized by glycerol, low temperature and the combination of glycerol and low temperature. The accurate quantitation of the protein by absorbance spectroscopy required exclusion of all contact with clarified polypropylene or polyvinyl chloride (PVC) materials. Although some of the fusion proteins were more prone to aggregation than the wild-type permease, the stability of a fusion protein containing a cytochrome(b562) insertion was indistinguishable from that of native lac permease.

Links

PubMed

Keywords

Chromatography, High Pressure Liquid; Cytochrome b Group/chemistry; Detergents; Drug Stability; Electrophoresis, Polyacrylamide Gel; Escherichia coli/chemistry; Escherichia coli/genetics; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Glycerol; Membrane Transport Proteins/chemistry; Membrane Transport Proteins/genetics; Monosaccharide Transport Proteins; Recombinant Fusion Proteins/chemistry; Recombinant Fusion Proteins/genetics; Solutions; Spectrophotometry, Ultraviolet; Symporters; Temperature

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