Production of Recombinant LysM-Fuison Proteins for Non-GMO Anchoring on Lactic Acid Bacteria

Abstract

Lactic acid bacteria (LAB) are traditionally used in the production and preservation of food and are natural inhabitants of the human gastrointestinal tract. This makes LAB generally regarded as safe for consumption. Some LAB exhibit probiotic properties and immunomodulatory effects, which contribute to their potential as ideal candidates as delivery vectors of immunogens. This study is part of a larger research project where the long-term goal is to develop mucosal vaccines by utilizing LAB as delivery vectors for antigens without genetically modifying the bacteria.

The aim of this thesis was to produce, purify, and anchor heterologous proteins to the cell wall of LAB using LysM-domains. In this work, two LysM-domains were used, one derived from Lactiplantibacillus pentosus KW1 and the other from L. pentosus KW2. These domains were fused N-terminally to the HaloTag reporter gene and the NetB W262A vaccine antigen. This antigen is a mutated form of the virulence factor NetB, produced by Clostridium perfringens, which causes necrotic enteritis in poultry. The fusion sequences were cloned into the inducible pBAD/HisB system and expressed in E. coli. Several attempts were made to optimize the production of soluble protein, including varying production conditions and the selection of production strain.

The fusion proteins were purified and bound to LAB under various binding conditions to evaluate their binding properties. Binding reactions were analyzed using SDS-PAGE and/or flow cytometry. Among the tested LAB, L. pentosus KW1 demonstrated the highest binding capacity to KW2_LysM_HaloTag, with minimal influence of temperature and reaction time on the amount of bound protein. However, different washing conditions for the bacterial cells prior to the binding reaction revealed that components in the MRS medium might affect the interaction between the LysM-domain and the peptidoglycan in the cell wall. To investigate the influence of cell wall components on the interaction, KW2_LysM_HaloTag was bound to peptidoglycan from Lactiplantibacillus plantarum WCFS1 and showed binding in PBS at pH 5.4. PBS buffers with varying pH were also used in the binding reactions and indicated that the interaction was pH dependent. The binding strength between the proteins and L. pentosus KW1 was evaluated by washing the bound protein with PBS, and the LysM-anchored proteins exhibited relatively stable binding. The characterization performed in this study demonstrated that the LysM-domain is essential for facilitating binding, and the binding of LysM-anchored proteins to L. pentosus KW1 highlights a promising potential for further development of a non-genetically modified LAB-based vaccine against necrotic enteritis in poultry.