Transport and anchoring of beta-lactamase to the external surface of Escherichia coli.

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 rust nine N-terminal amino acids of the mature major Escherichia coli lipoprotein, (it) amino acids 46-159 ofthe outer membrane protein OmpA, and (Mi) the complete mature ,-lactamase (EC 3.5.2.6) sequence. This protein had an enzymatically active ,-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 ,t-lactamase domain of the fusion protein was exposed on the external surface of E. coil. In cells grown at 240C the localization of 8-lactamase on the cell surface was almost quantitative (>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, 13-lactamase, can be transported through-and become anchored on-the outer membrane by fusion to the proper targeting and localization signals.

Amperometric l-glutamate biosensor based on bacterial cell-surface displayed glutamate dehydrogenase

“…Many studies showed that enzyme could be displayed on the cell surface of bacteria and retain its relative spatial structure and biological activity [42,43]. Gldh is widely existed in various living organisms and involved in amino acid metabolism [44].…”

Biosynthetic lipid‐tagging of antibodies

“…Francisco and coworkers recently demonstrated that a fusion construct which contains the lipoprotein signal sequence and nine N-terminal amino acids followed by amino acids 45-159 of the outer membrane protein OmpA can be used to express single-chain antibodies and other proteins on the bacterial cell surface [22,271. The lipoprotein part targets the protein to the outer membrane whereas the transmembrane OmpA part is used to obtain the correct topological orientation of the protein on the outer surface [22,27].…”

“…The lipoprotein part targets the protein to the outer membrane whereas the transmembrane OmpA part is used to obtain the correct topological orientation of the protein on the outer surface [22,27]. While this approach may prove useful in the screening of surface displayed antibodies and in catalytic and other biotechnological applications (e.g.…”

Development of a novel mammalian cell surface antibody display platform

“…During the past decade, antibody display technology has become increasingly valuable for the development of antibody therapeutics. Several cell-based display techniques have been developed including phage display, [3][4][5][6][7][8] E. coli display [9][10][11] and more recently, yeast display. [12][13][14][15][16][17] Each display system has its strengths and weaknesses.…”