Expression of recombinant Rhipicephalus appendiculatus Ra85 gut protein in Pichia pastoris yeast cells

Abstract

Rhipicephalus appendiculatus is a major vector responsible for transmitting Theileria parva, the causative agent of East Coast Fever in cattle across sub-Saharan Africa. The control of this tick species has traditionally relied on acaricides, which pose risks of resistance development and environmental contamination. As a sustainable alternative, the development of anti-tick vaccines targeting tick antigens such as Ra85 has gained attention. However, successful production of immunogenic proteins in sufficient quantity and quality remains a critical challenge. This study explored the expression of the recombinant Ra85 protein in Pichia pastoris, a eukaryotic expression system known for efficient protein processing and secretion. The main objectives of the study were to determine the optimal expression conditions of Ra85 protein and to express the recombinant protein in the Pichia pastoris GS115 strain. Plasmid DNA containing the Ra85 gene was first extracted from E. coli Top10 F’ cells and then linearized to facilitate genomic integration into the P. pastoris expression system. Following successful transformation, expression trials were conducted under varying conditions to determine the optimal temperature and induction time. Expression was monitored through visual assessment of protein bands via gel electrophoresis. The results showed successful extraction and linearization of the plasmid DNA, indicating that the vector was suitable for transformation. The optimal expression conditions for Ra85 protein in P. pastoris were found to be at a temperature range of 28–30°C and an induction period of three days, with cultures maintained in an incubator shaker at 200 rpm. These conditions allowed for visible expression of Ra85, suggesting that the Pichia pastoris system can effectively produce the protein. While high RNA contamination was initially observed during plasmid extraction, it did not impede downstream processes. In conclusion, this study demonstrated that Pichia pastoris is a viable host for the expression of Ra85, offering a promising platform for the production of candidate vaccine antigens. The optimized expression conditions identified in this study can support future efforts in protein purification, immunogenicity testing, and large-scale production. These findings contribute to the growing body of research aimed at developing effective anti-tick vaccines as a sustainable alternative to chemical control methods.