Heterologous expression of Cysteine Protease 8 from Trichomonas foetus in Pichia pastoris
Karli Geethanjali , Polavarapu Rathnagiri and Varada Kalarani
Journal of Veterinary and Animal Sciences.2023.54(1):13-20
Karli Geethanjali : Asst.Professor, Dept. of Biotechnology, Indira Priyadarshini Govt. Degree College for Women, Nampally, Hyderabad, Telangana, INDIA & Research scholar, Dept. of Biotechnology, SPMVV, Tirupati, INDIA.
Polavarapu Rathnagiri : CEO &M D, Genomix Biotech Inc 2620 Braithwood Road, Atlanta, GA 30345; USA and Genomix, Molecular diagnostics, Hyderabad, INDIA.
Varada Kalarani : CEO, Women Biotech incubation facility (DBT-Bio-Nest) & Rtd. Professor and Head, Dept. of Biotechnology, SPMVV, Tirupati, INDIA
Received: 04.05.2022 Accepted: 20.10.2022 Published online: 31.03.2023
Bovine trichomonosis is one of the most neglected venereal diseases of cattle. Trichomonas foetus, the causative organism was known over decades and is responsible for severe reproductive failure. Except for a few lab-based assays, to date, there are no point-of-care diagnostics developed to screen for the presence of infectious agents in cattle. In this study, we have identified cysteine protease 8 as a suitable antigenic protein for developing sero-diagnostics. A 960 bp Tf CP8 gene was cloned into methylotrophic Pichia pastoris X-33 by homologous recombination using a pPICZαA vector for recombinant protein expression. The traditional fed-batch method of induction with methanol resulted in inconsistent expression in 48h incubation, hence a novel single batch culture with 1% methanol induction for 24h was standardised and obtained optimal recovery of approximately 36 KDa recombinant protein secreted into media. To the best of our knowledge, this is the first report of cloning and expression of genes from Trichomonas foetus. This CP8 protein could be further optimised for developing lateral flow assays and ELISA as point-of-care tools.
Keywords: Fed-batch culture, His Tag, methylotrophic yeast