Stability of the lactose permease in detergent solutions.
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.
Engel
Christian K
CK
Division of Molecular and Structural Biology, Ontario Cancer Institute, Toronto, Canada.
Chen
Lu
L
Privé
Gilbert G
GG
D002851:Chromatography, High Pressure Liquid; D003573:Cytochrome b Group / Q000737:chemistry; D003902:Detergents; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D005990:Glycerol; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D012996:Solutions; D013056:Spectrophotometry, Ultraviolet; D027981:Symporters; D013696:Temperature
D003573:Cytochrome b Group; D003902:Detergents; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; D012996:Solutions; D027981:Symporters; C017850:cytochrome b562, E coli; C021499:lactose permease; D005990:Glycerol