Novel Surface Display System for Proteins on Non-Genetically Modified Gram-Positive Bacteria

Abstract

A novel display system is described that allows highly efficient immobilization of heterologous proteins on bacterial surfaces in applications for which the use of genetically modified bacteria is less desirable. This system is based on nonliving and non-genetically modified gram-positive bacterial cells, designated grampositive enhancer matrix (GEM) particles, which are used as substrates to bind externally added heterologous proteins by means of a high-affinity binding domain. This binding domain, the protein anchor (PA), was derived from the Lactococcus lactis peptidoglycan hydrolase AcmA. GEM particles were typically prepared from the innocuous bacterium L. lactis, and various parameters for the optimal preparation of GEM particles and binding of PA fusion proteins were determined. The versatility and flexibility of the display and delivery technology were demonstrated by investigating enzyme immobilization and nasal vaccine applications.

Mucosal vaccine delivery of antigens tightly bound to an adjuvant particle made from food-grade bacteria

“…Binding of the PA to GEM particles is non-covalent, but very strong and approximately one million PA molecules can be bound on to one particle [31]. So far, only treatment with SDS sample buVer or 8 M LiCl was able to partially release the PA (patent WO02101026 [19]).…”

“…Bosma et al [31] have shown the versatility and Xexibility of this system by functional display of two enzymes, -amylase and -lactamase, in diVerent ratios onto the surface of GEM particles. PA-mediated surface display diVers in a number of aspects from bacterial surface display and delivery systems known to date [20].…”

Peptidoglycan N-acetylglucosamine deacetylation decreases autolysis in Lactococcus lactis

“…AcmA consists of two domains: an active site domain and a C-terminal region containing three highly homologous repeats of 45 aa, named LysM domains, which bind peptidoglycan (Steen et al, 2003). To study the binding of AcmA to peptidoglycan with different degrees of de-Nacetylation, we used a fusion protein consisting of the Cterminal peptidoglycan-binding domain of AcmA fused to a c-Myc epitope (c-Myc-PA) at its N terminus (Bosma et al, 2006). Peptidoglycan extracted from IL6288, pgdA mutant TIL926 and pgdA-overexpressing strain TIL926(pJIMpgdA) was incubated with different concentrations of c-Myc-PA.…”

“…The cMyc-PA (PA3) fusion protein corresponds to the cell wall binding domain of AcmA (PA; C-terminal 218 aa of AcmA) fused to a c-Myc epitope at its N terminus (Bosma et al, 2006). It was produced in the supernatant of strain L. lactis PA1001(pPA3) (a kind gift of K. Leenhouts, BioMade, Groningen, The Netherlands).…”

Versatile microbial surface-display for environmental remediation and biofuels production

“…Another approach is the use of chemically pretreated and boiled L. lactis as a matrix to bind heterologous proteins externally [65]. The protein peptidoglycan hydrolase, AcmA, was demonstrated to display α-amylase and β-lactamase, as well as epitopes of the Plasmodium berghei malaria circumsporozoite protein antigen.…”