Laboratory Scale Production of Recombinant Haa86 Tick Protein in Pichia pastoris and in Escherichia coli System
The commercial recombinant Bm86-based vaccines against Rhipicephalus (Boophilus) microplus in Australia (TickGARD™, TickGARD plus™) and in Cuba (Gavac™) provided significant impetus to researchers globally to work on anti-tick vaccines. The Bm86 homologue
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Introduction Hyalomma anatolicum, a tick vector for Theileria annulata responsible for bovine tropical theileriosis, is prevalent in many parts of the world and almost all over India and causes heavy economic loss to livestock sector [1]. As an alternative to acaricide, the immunological control of ticks was found to be an effective component of the integrated control of the tick species [2]. In the line of success of Bm86-based vaccines against Rhipicephalus (Boophilus) microplus (TickGARD™, TickGARD plus™, and Gavac™) [3–5], the Bm86 homologue of H. anatolicum was expressed in both prokaryotic and eukaryotic expression systems, and its efficacy against both homologous and heterologous challenge were recorded [6, 7]. Purification of native midgut antigen from ticks is tedious, laborious, time-consuming, and low-yielding procedure. Immunization trials in large animals followed by commercialization of vaccines necessitate the production of antigens in bulk quantities. Recombinant DNA technology using prokaryotic or eukaryotic expression systems have been utilized for the generation of targeted proteins in bulk. Both the above systems have certain advantage and disadvantage over each other (Table 1). Yeast offers site-specific integration, increase in copy number, leader sequence for the secretion of heterologous protein, posttranslational modifications, fast growth, and low-cost media [8, 9]. Similarly, E. colibased expression system is well known for its simplicity, flexibility, and inexpensive expression of target protein. Moreover, extensive information of genetics and vast availability of compatible tools for genetic manipulation makes the system very popular [10].
Sunil Thomas (ed.), Vaccine Design: Methods and Protocols, Volume 2: Vaccines for Veterinary Diseases, Methods in Molecular Biology, vol. 1404, DOI 10.1007/978-1-4939-3389-1_30, © Springer Science+Business Media New York 2016
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Table 1 Advantages and disadvantages of prokaryotic and eukaryotic expression system Prokaryotic expression system
Eukaryotic expression system
Advantages
Advantages
•
Easy cloning and genetic manipulation
•
Post-translational modifications are possible
•
Inexpensive to culture
•
Protein secreted in medium can easily be purified
•
Rapid growth and fast expression
•
Rapid growth and high expression
• Flexible in expression (multiple promoters, tags, fusion proteins, cleavage site, etc.) • Usually work well for intracellular proteins •
Can be optimized for soluble expression vs inclusion bodies
Disadvantages
Disadvantages
•
Unavailability of eukaryotic post-translational modifications
•
•
Lack of some tRNA common to eukaryotic genes which severely limits the expression and necessitates codon optimization of the eukaryotic gene to be expressed
• Inability to perform certain complex posttranslational modifications, such as prolyl hydroxylation and amidation as well as some types of phosphorylation and glycosylation
•
Difficult to express the gene of interest as secretory
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