Recombinant protein immobilisation and display by alginate : a thesis presented in partial fulfilment of the requirements of the degree of Master of Science in Microbiology at Massey University, Palmerston North, New Zealand. EMBARGOED until 6 May 2018
Biopolymers are a diverse group of organic materials with important applications in a number of industries. Their ability to adsorb and encapsulate compounds has been widely utilised in both biotechnologies and pharmaceuticals. In the last decade, biopolymers have been given new and enhanced functionality, including the separation and purification of compounds. This field is of increasing relevance as advances in the bacterial cell culture process have improved productivity in the biomanufacturing industry, with the establishment of several bacterial host cell lines and optimised protein production systems. This increase in upstream productivity is leading to bottlenecks in downstream processing as current technology platforms reach their limits of throughput and scalability. While previous studies have generated functionalised protein biopolymers using polyhydroxyalkanoate (PHA) biopolyester beads, very few studies have examined the commercially significant biopolymer alginate. Alginate is an exopolysaccharide produced by algae and some bacteria, and is widely utilised in food, pharmaceutical, and biomedical industries because of its stabilising, haemostatic, biocompatible properties and its modifiable structure. In this study, a partially functional alginate-binding recombinant protein was produced, which contained an α-amylase domain from Bacillus licheniformis (BLA) translationally fused to the alginate-binding domain of Pseudomonas aeruginosa AlgX – an alginate acetyltransferase. An Ssp DnaB mini-intein was included between BLA and AlgX to facilitate recovery of BLA, following immobilisation and display on the surface of alginate. However, aberrant activity of the intein caused total cleavage of the recombinant protein between its BLA and AlgX domains before it could be recovered from the protein production system. Additionally, the absence of a key cysteine residue in the alginate-binding domain prevented the formation of a disulfide bond, which is an essential structural element for the folding and functionality of this region. While this study was unable to overcome intein hyperactivity, functional analysis of the BLA domain showed consistent and significant levels of α-amylase activity, leading to a positive outlook for the functionality of a full-length recombinant protein if proper intein activity can be restored and the necessary cysteine included. In this way, alginate could be specifically functionalised with a desired protein, and in turn, alginate beads could be used for the separation and enrichment of target proteins.