RESUMO
OBJECTIVE: To develop a deliberately engineered expression and purification system for an active chimeric-recombinant tissue plasminogen activator (crtPA) using co-expression with polyhydroxybutyrate (PHB) operon genes. RESULTS: Fusion of crtPA with PhaC-synthase simplified the purification steps through crtPA sedimentation with PHB particles. Moreover, the covalently immobilized crtPA was biologically active as shown in a chromogenic assay. Upon WELQut-protease activity, the released single-chain crtPA converted to the two-chain form which produced a pattern of bands with approx. MW of 32 and 11 kDa in addition to the full length crtPA. CONCLUSION: Fusion of crtPA with PhaC-synthase not only simplifies purification from the bacterial host lysate, but also co-expression of PHB operon genes creates an oxidative environment, thereby reducing the inclusion body formation possibility. The isolated crtPA-PHB granules exhibited crtPA serine protease activity. Thus, fusion with the PhaC protein could be used as a scaffold for covalent displaying of functional disulfide-rich proteins.
Assuntos
Aciltransferases/metabolismo , Hidroxibutiratos/química , Hidroxibutiratos/metabolismo , Ativador de Plasminogênio Tecidual/genética , Aciltransferases/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cupriavidus necator/genética , Cupriavidus necator/metabolismo , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Propriedades de Superfície , Ativador de Plasminogênio Tecidual/biossíntese , Ativador de Plasminogênio Tecidual/metabolismoRESUMO
Heterogeneity of subgenomic regions of hepatitis C virus (HCV) may be associated with response to interferon (IFN) therapy. The amino acid sequences of the PKR/eIF-2α phosphorylation homology domain (pePHD), IFN sensitivity determining region (ISDR), PKR binding domain (PKRBD), and variable region 3 (V3) were studied in 19 patients before and after 4 weeks of treatment. All patients were infected with HCV genotype 1a and were treated with pegylated-IFN and ribavirin. Thirteen patients achieved sustained viral response (responders) and six failed to clear viral RNA (nonresponders). The amino acid sequences in the pePHD and ISDR were identical in responders and nonresponders. However, amino acid substitution at position 2252 of PKRBD was significantly different between responders and nonresponders (P = 0.044). A larger number of mutations were observed in the V3 region of responders (P < 0.001). In this region, the amino acid in position 2364 differed between responders and nonresponders (responders: aspartic acid and serine, nonresponders: asparagine, P = 0.018). The amino acid sequences in the regions which were studied did not change after 4 weeks of treatment. It is concluded that the presence of specific amino acids in position 2252 of PKRBD and position 2364 of V3 might be associated with clinical response to IFN.
