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Cumbassa A.,Instituto Nacional Of Investigacao Agraria E Veterinaria Iniav | Barahona M.J.,Instituto Nacional Of Investigacao Agraria E Veterinaria Iniav | Cunha M.V.,Instituto Nacional Of Investigacao Agraria E Veterinaria Iniav | Cunha M.V.,University of Lisbon | And 6 more authors.
Veterinary Microbiology | Year: 2015

Coxiella burnetii is the etiological agent of Q fever or Coxiellosis, a zoonosis mainly affecting domestic ruminants. Information on the population structure and epidemiology of C. burnetii in animals is scarce in Portugal. Evidence of C. burnetti infection was sought in domestic, wild and captive animals based on the detection of bacterial DNA. Tissue samples from 152 domestic animals (cattle = 24, goats = 51, sheep = 76 and swine = 1), 55 wild carnivores (Egyptian mongoose = 45, red fox = 4, common genet = 3, weasel = 2 and European badger = 1) and 22 zoo animals (antelopes = 15, impala = 1; rhinoceros = 1, deer = 2, zebras = 2 and giraffe = 1) were screened by nested-touchdown PCR. Cloacae swabs from 19 griffon vultures were also analysed. Among the domestic ruminants, goats presented the highest prevalence of infection (23.53%), followed by cattle, (20.83%) and sheep (10.53%). C. burnetii DNA was also detected in five Egyptian mongooses and two antelopes and one giraffe. Using a 6-locus multiple-locus variable-number tandem repeat analysis (MLVA-6) six complete genotypes, T, I and CM and the first reported CN, CO and CP, were identified, respectively, in small ruminants and Egyptian mongooses. Clustering analysis of genotypes exposed four distinct groups, according to detection source, enlightening an apparent association between C. burnetii genotype and host. © 2015 Elsevier B.V.

Costa S.J.,University of Minho | Costa S.J.,Instituto Nacional Of Saude Dr Ricardo Jorge Insarj | Coelho E.,University of Minho | Coelho E.,Instituto Nacional Of Saude Dr Ricardo Jorge Insarj | And 6 more authors.
Protein Expression and Purification | Year: 2013

Downstream processing is still a major bottleneck in recombinant protein production representing most of its costs. Hence, there is a continuing demand of novel and cost-effective purification processes aiming at the recovery of pure and active target protein. In this work, a novel purification methodology is presented, using the Fh8 solubility enhancer tag as fusion handle. The binding properties of Fh8 tag to a hydrophobic matrix were first studied via hydrophobic interaction chromatography (HIC). The Fh8 tag was then evaluated as a purification handle by its fusion to green fluorescent protein and superoxide dismutase. The purification efficiency of the Fh8-HIC strategy was compared to the immobilized metal ion affinity chromatography (IMAC) using the His 6 tag. Results showed that the Fh8-HIC binding mechanism is calcium-dependent in a low salt medium, making the purification process highly selective. Both target proteins were biologically active, even when fused to Fh8, and were successfully purified by HIC, achieving efficiencies identical to those of IMAC. Thus, the Fh8 acts as an effective affinity tag that, together with its previously reported solubility enhancer capability, allows for the design of inexpensive and successful recombinant protein production processes in Escherichia coli. © 2013 Published by Elsevier Inc.

Costa S.J.,IBB Institute for Biotechnology And Bioengineering | Costa S.J.,Instituto Nacional Of Saude Dr Ricardo Jorge Insarj | Almeida A.,Hitag Biotechnology Lda. | Almeida A.,University of Porto | And 3 more authors.
Applied Microbiology and Biotechnology | Year: 2013

The Escherichia coli host system is an advantageous choice for simple and inexpensive recombinant protein production but it still presents bottlenecks at expressing soluble proteins from other organisms. Several efforts have been taken to overcome E. coli limitations, including the use of fusion partners that improve protein expression and solubility. New fusion technologies are emerging to complement the traditional solutions. This work evaluates two novel fusion partners, the Fh8 tag (8 kDa) and the H tag (1 kDa), as solubility enhancing tags in E. coli and their comparison to commonly used fusion partners. A broad range comparison was conducted in a small-scale screening and subsequently scaled-up. Six difficult-to-express target proteins (RVS167, SPO14, YPK1, YPK2, Frutalin and CP12) were fused to eight fusion tags (His, Trx, GST, MBP, NusA, SUMO, H and Fh8). The resulting protein expression and solubility levels were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis before and after protein purification and after tag removal. The Fh8 partner improved protein expression and solubility as the well-known Trx, NusA or MBP fusion partners. The H partner did not function as a solubility tag. Cleaved proteins from Fh8 fusions were soluble and obtained in similar or higher amounts than proteins from the cleavage of other partners as Trx, NusA or MBP. The Fh8 fusion tag therefore acts as an effective solubility enhancer, and its low molecular weight potentially gives it an advantage over larger solubility tags by offering a more reliable assessment of the target protein solubility when expressed as a fusion protein. © 2012 Springer-Verlag Berlin Heidelberg.

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