Fixation and stabilization of Escherichia coli cells displaying genetically engineered cell surface proteins

A large biotechnological potential is inherent in the display of proteins (e.g., enzymes, single-chain antibodies, on the surface of bacterial cells) (Georgiou et al., 1993). Applications such as immobilized whole-cell biocatalysts or cellular adsorbents require cell fixation to prevent disintegration, stabilization of the anchored protein from leakage, denaturation or proteolysis, and total loss of cell viability, preventing medium and potential product contamination with cells. In this article we describe the adaptation of a simple two-stage chemical crosslinking procedure based on 'bi-layer encagement' (Tor et al., 1989) for stabilizing Escherichia coli cells expressing an Lpp-OmpA (46-159)-β-lactamase fusion that displays β-1actamase on the cell surface. Bilayer crosslinking and coating the bacteria with a polymeric matrix is accomplished by treating the cells first with either glutaraldehyde or polyglutaraldehyde, followed by secondary crosslinking with polyacrylamide hydrazide. These treatments resulted in a 5- to 25 fold reduction of the thermal inactivation rate constant at 55°C of surface anchored β-lactamase and completely prevented the deterioration of the cells for at least a week of storage at 4°C. The stabilization procedure developed paves the way to scalable biotechnological applications of E. coli displaying surface anchored proteins as whole-cell biocatalysts and adsorbents.