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In Vivo Labeling of Escherichia Coli Cell Envelope Proteins With N-hydroxysuccinimide Esters of Biotin

In Vivo Labeling of Escherichia Coli Cell Envelope Proteins With N-hydroxysuccinimide Esters of Biotin

J A Bradburne, P Godfrey, J H Choi, J N Mathis

Appl Environ Microbiol. 1993 Mar;59(3):663-8.

PMID: 8480997

Abstract:

The primary amine coupling reagents succinimidyl-6-biotinamido-hexanoate (NHS-A-biotin) and sulfosuccinimidyl-6-biotinamido-hexanoate (NHS-LC-biotin) were tested for their ability to selectively label Escherichia coli cell envelope proteins in vivo. Probe localization was determined by examining membrane, periplasmic, and cytosolic protein fractions. Both hydrophobic NHS-A-biotin and hydrophilic NHS-LC-biotin were shown to preferentially label outer membrane, periplasmic, and inner membrane proteins. NHS-A- and NHS-LC-biotin were also shown to label a specific inner membrane marker protein (Tet-LacZ). Both probes, however, failed to label a cytosolic marker (the omega fragment of beta-galactosidase). The labeling procedure was also used to label E. coli cells grown in low-salt Luria broth medium supplemented with 0, 10, and 20% sucrose. Outer membrane protein A (OmpA) and OmpC were labeled by both NHS-A- and NHS-LC-biotin at all three sucrose concentrations. In contrast, OmpF was labeled by NHS-A-biotin but not by NHS-LC-biotin in media containing 0 and 10% sucrose. OmpF was not labeled by either NHS-A- or NHS-LC-biotin in E. coli cells grown in medium containing 20% sucrose. Coomassie-stained gels, however, revealed similar quantities of OmpF in E. coli cells grown at all three sucrose concentrations. These data indicate that there was a change in outer membrane structure due to increased osmolarity, which limits accessibility of NHS-A-biotin to OmpF.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR42415182	β-Galactosidase-biotin labeled from Escherichia coli			Price

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