Production and biochemical characterization of the recombinant Boophilus microplus Bm95 antigen from Pichia pastoris

Abstract

The new antigen Bm95 from the cattle tick Boophilus microplus was recently isolated, cloned and expressed in the methylotrophic yeast Pichia pastoris. The recombinant protein has shown to induce protection in cattle against infestations of B. microplus under controlled and production conditions. In this paper we report the production and large-scale purification of the Bm95 protein, following a simple and cost-effective process. The antigen was obtained highly aggregated, forming particles ranging from 26 to 30 nm and with purity higher than 80%. The process yield was 0.55 g of pure Bm95 protein per liter of culture. The 98% of the primary structure of the recombinant protein was verified by mass spectrometry. Three amino acid changes in comparison with the sequence deduced from cDNA were detected by LC-MS/MS. The antigen was also obtained N-glycosylated, as previously reported for heterologous protein expression in P. pastoris.

Expression of recombinant Rhipicephalus (Boophilus) microplus, R. annulatus and R. decoloratus Bm86 orthologs as secreted proteins in Pichia pastoris

“…The recombinant Bm86 has been expressed in Escherichia coli [ 10 ], Aspergillus nidulans and A. niger [ 23 ] and Pichia pastoris [ 11 , 24 , 25 ]. Of these expression systems, P. pastoris has been shown to be the more efficient for protein secretion [ 26 , 27 ].…”

“…Furthermore, production of Bm86 in P. pastoris may increase the antigenicity and immunogenicity of the recombinant antigen [ 28 , 29 ]. However, the process previously reported for the production of recombinant Bm86 in P. pastoris is not based on protein secretion but on the expression of the antigen anchored to the yeast membrane, making necessary the purification under denaturing conditions followed by refolding of an antigen with high number of disulfide bonds [ 24 , 25 , 30 ]. Recently, R. decoloratus Bm86 orthologs were cloned, expressed in E. coli and partially characterized [ 31 ].…”

Expressão de Xilanases de Aspergillus nidulans em Pichia pastoris: Purificação, Caracterização e Aplicação na Hidrólise do Bagaço de Cana-de-Açúcar

“…A enzima álcool oxidase (AOX, EC 1.1.3.13) catalisa o primeiro passo da via de utilização do metanol (via MUT) (Bobrowicz et al, 2004). O genoma de Pichia pastoris contém dois genes AOX: AOX1 compreende 30% das proteínas solúveis totais no extrato de Pichia pastoris crescida somente em metanol e é controlada pelo promotor forte pAOX1 (Bobrowicz et al, 2004;Bos et al, 2003;Boue et al, 2004); AOX2 é responsável por 15% da atividade AOX total da célula e é controlado pelo promotor mais fraco pAOX2 (Boysen et al, 2004). Ambos os promotores são induzidos por metanol.…”

Anti-tick Vaccines for the Control of Ticks Affecting Livestock

“…pastoris [ 48 ] and incorporated in a number of vaccine trials. Effects of Bm95 vaccination are similar to Bm86 vaccination with tick rejection, damage, and death.…”