PMID:1557377
| Citation |
Francisco, JA, Earhart, CF and Georgiou, G (1992) Transport and anchoring of beta-lactamase to the external surface of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 89:2713-7 |
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| Abstract |
The outer membrane of Gram-negative bacteria presents an effective barrier that restricts the release of proteins from the cell. Virtually all extracellular proteins of Gram-negative bacteria are exported by specialized systems requiring the action of several gene products. We have constructed a tripartite fusion consisting of (i) the signal sequence and first nine N-terminal amino acids of the mature major Escherichia coli lipoprotein, (ii) amino acids 46-159 of the outer membrane protein OmpA, and (iii) the complete mature beta-lactamase (EC 3.5.2.6) sequence. This protein had an enzymatically active beta-lactamase and was found predominantly in the outer membrane. Immunofluorescence microscopy, the accessibility of the fusion protein to externally added proteases, and the rates of hydrolysis of nitrocefin and penicillin G by whole cells demonstrated that a substantial fraction (20-30%) of the beta-lactamase domain of the fusion protein was exposed on the external surface of E. coli. In cells grown at 24 degrees C the localization of beta-lactamase on the cell surface was almost quantitative (greater than 80% of the enzymatically active protein was exposed to the extracellular fluid) as determined by nitrocefin and penicillin G hydrolysis and trypsin accessibility. These results demonstrated that a soluble protein, beta-lactamase, can be transported through--and become anchored on--the outer membrane by fusion to the proper targeting and localization signals. |
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| Keywords |
Amino Acid Sequence; Bacterial Outer Membrane Proteins/chemistry; Bacterial Outer Membrane Proteins/metabolism; Bacterial Proteins/metabolism; Biological Transport; Cell Compartmentation; Escherichia coli/metabolism; Genetic Engineering; Lipoproteins/metabolism; Molecular Sequence Data; Recombinant Fusion Proteins/metabolism; Structure-Activity Relationship; beta-Lactamases/chemistry; beta-Lactamases/metabolism |
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