FIXATION AND STABILIZATION OF ESCHERICHIA-COLI-CELLS DISPLAYING GENETICALLY-ENGINEERED CELL-SURFACE PROTEINS

A large biotechnological potential is inherent in the display of prote ins (e.g., enzymes, single-chain antibodies, on the surface of bacteri al cells) (Georgiou et al., 1993). Applications such as immobilized wh ole-cell biocatalysts or cellular adsorbents require cell fixation to prevent disintegration, stabilization of the anchored protein from lea kage, denaturation or proteolysis, and total loss of cell viability, p reventing medium and potential product contamination with cells. In th is article we describe the adaptation of a simple two-stage chemical c rosslinking procedure based on ''bi-layer encagement'' (Tor et al., 19 89) for stabilizing Escherichia coli cells expressing an Lpp-OmpA (46- 159)-beta-lactamase fusion that displays beta-lactamase on the cell su rface. Bilayer crosslinking and coating the bacteria with a polymeric matrix is accomplished by treating the cells first with either glutara ldehyde or polyglutaraldehyde, followed by secondary crosslinking with polyacrylamide hydrazide. These treatments resulted in a 5- to 25-fol d reduction of the thermal inactivation rate constant at 55 degrees C of surface anchored beta-lactamase and completely prevented the deteri oration of the cells for at least a week of storage at 4 degrees C. Th e stabilization procedure developed paves the way to scalable biotechn ological applications of E. coil displaying surface anchored proteins as whole-cell biocatalysts and adsorbents. (C) 1996 John Wiley & Sons, Inc.