Computational protocol: Protective Antigens Against Glanders Identified by Expression Library Immunization

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Protocol publication

[…] Cloning and protein expression protocols were followed as described previously (Whitlock et al., ). Briefly, target sequences were subjected to bioinformatics analysis using SignalP v.3.0 (Bendtsen et al., ), TMHMM v.2.0 (Krogh et al., ), and PHYRE v.0.2 (Kelley et al., ) to identify putative N-terminal amino acid (AA) secretion sequences, transmembrane domains, and homologies to published crystal structures, respectively. Protein subcellular localization was predicted using PSORTb v.3.0.2 (Yu et al., ) and CELLO v.2.5 (Yu et al., ). Informatically predicted signal peptides and transmembrane sequences were identified so as to avoid them if possible in the design of the ORFs for cloning into bacterial expression constructs. This was intended to facilitate recombinant production. The redesigned ORFs were PCR-amplified from genomic B. mallei DNA and cloned into pET28a(+) (Novagen), or pcDNA3.1 (Invitrogen) expression vector, in frame with either an N- or C-terminal 6× His affinity tag, or both. Oligo primers introducing specific restriction enzyme sites were purchased from Integrated DNA Technologies. In addition to the new targets, the GroEL gene (BMA_2001) was cloned to be produced and purified for use as a previously characterized immunogen (Amemiya et al., ).For target protein production, E. coli host Rosetta (λDE3) was transformed with the expression constructs. Expression was induced by growth in Overnight Express instant TB medium (Novagen) for 18–20 h. Bacterial pellets were lysed using 10× CelLytic B (Sigma), and 6× His-tagged proteins purified by Ni2+ affinity chromatography. Purified proteins were dialyzed against two changes of 10 mM HEPES/150 mM NaCl, pH 7.4, aliquoted and stored at −80°C. Protein concentrations were determined using the BCA kit (Pierce) using bovine serum albumin (BSA) as a standard. Five of the 12 candidates and the previously identified antigen GroEL (hsp60) protein were sufficiently produced, with either an N- or C-terminal tag, or both, for vaccine testing. BMA_2001/GroEL (residues 1–550) and BMA_2821 (residues 418–753) were produced with both N- and C-terminal His tags. BMA_2804 (residues 17–370), BMA_A0768 (residues 20–489), and BMA_0816 were produced with C-terminal tags. BMA_0816 was produced in three sub-fragments (residues 43–334, 35–668, and 669–930) with C-terminal tags. […]

Pipeline specifications

Software tools SignalP, TMHMM, Phyre, PSORTb
Applications Protein sequence analysis, Membrane protein analysis
Organisms Burkholderia mallei ATCC 23344, Homo sapiens, Burkholderia mallei, Burkholderia pseudomallei, Mus musculus
Diseases Diabetes Mellitus, Lung Diseases, Neoplasms, Opportunistic Infections