No description available.

Staphylococcus aureus secretes two cysteine proteases, Staphopain A (scpA) and Staphopain B (sspB). We hypothesized that ScpA will exhibit a distinct activation mechanism, and a different or complementary substrate specificity compared to SspB. A Cys>Ala active site substitution led to the accumulation of unprocessed 40-kDa proScpA, confirming that ScpA undergoes autocatalytic activation. A temporal analysis of ScpA expression revealed that activation was initiated by processing at Lys171 and Glu176, producing an intermediate that was rapidly converted to several isoforms of mature protease by processing after Thr202, Lys209, Thr214 and Asn216. Consistent with broad specificity, mature ScpA was sensitive to autocatalytic degradation. ScpA demonstrated activity towards elastin, fibrinogen and indicated evidence for binding to heparin. Elastinolytic activity was uniquely associated with strains belonging to CC30, and was correlated with ScpA expression. Therefore, although ScpA and SspB share both sequence and structural similarity, they exhibited very different substrate specificities and activation mechanisms.

Staphylococcus aureus secretes two cysteine proteases, Staphopain A (scpA) and Staphopain B (sspB). We hypothesized that ScpA will exhibit a distinct activation mechanism, and a different or complementary substrate specificity compared to SspB. A Cys>Ala active site substitution led to the accumulation of unprocessed 40-kDa proScpA, confirming that ScpA undergoes autocatalytic activation. A temporal analysis of ScpA expression revealed that activation was initiated by processing at Lys171 and Glu176, producing an intermediate that was rapidly converted to several isoforms of mature protease by processing after Thr202, Lys209, Thr214 and Asn216. Consistent with broad specificity, mature ScpA was sensitive to autocatalytic degradation. ScpA demonstrated activity towards elastin, fibrinogen and indicated evidence for binding to heparin. Elastinolytic activity was uniquely associated with strains belonging to CC30, and was correlated with ScpA expression. Therefore, although ScpA and SspB share both sequence and structural similarity, they exhibited very different substrate specificities and activation mechanisms.

Serine protease inhibitors (serpins) are a large superfamily of proteins that regulate critical proteolytic pathways by inhibiting serine proteases. Tick-encoded serpins are thought to play a vital role in the feeding process. To determine the relationship of Amblyomma americanum serpin 6 (S6) to tick feeding regulation, this study attempted to define the biological significance of this molecule through transcription and protein expression profiles, biochemical characterization of recombinant s6 (rS6), and the effects of in vivo post-transcriptional gene silencing on blood meal acquisition and fecundity. Transcriptional analysis revealed that S6 mRNA is ubiquitously expressed in unfed and partially fed ticks through the initial 5 days of the feeding period. S6 mRNA abundance in dissected tick organs showed a 3.7, 3.4, and 1.7- fold upregulation from 24 h to 96 h in the salivary gland (SG), midgut (MG) and the carcass (CA) remnant after removal of SG, MG respectively before downregulating at 120 h. Native S6 protein is downregulated in response to tick feeding, with correlation between transcription and protein expression profiles only consistent from the unfed to 48 h. Similarly, S6 protein expression in dissected female tick tissues is reduced as feeding progresses, with S6 being identified in SG, MG, ovary (OV), and CA from 24 h until 72 h. Biochemical characterization of S6 was not achieved, as rS6 did not form an irreversible complex when incubated with chymotrypsin or trypsin. Although complete silencing of S6 and S6/S17 mRNA was achieved, post-transcriptional gene knockdown had no effect on tick feeding efficiency or fecundity. These findings have been discussed in regards to the development of a vaccine against A. americanum and necessary future studies have been suggested for further characterization and assessment of biological significance.

Taura syndrome virus (TSV), the epidemic agent of Taura syndrome (TS), was tentatively classified in the family Dicistroviridae, caused disastrous losses with high mortality rates from 60 to >90% of affected pond-cultured Litopenaeus vannamei. The amino acid sequence alignments of ORF1 revealed sequence motifs characteristic of a helicase, a protease and an RNA-dependent RNA polymerase, similar to the non-structural proteins of picornavirus, suggesting perhaps a similar to picornavirus polyprotein processing. Proteolytic processing of the TSV polyprotein and protease is still unclear. In order to further understand the role of protease interaction with polyprotein, several clones were constructed. In this study, we constructed several clones that expressed the TSV virus coat protein(VP-polyprotein)¡]pTSVVP¡^, putative 3C protease¡]pTSV3C¡^, protease¡ÏRdRp-polyprotein(pET3CD) and both VP+3C proteins(pTSVVP3C) in E. coli. The VP-polyprotein band was excised from SDS-polyacrylamide gels and electroeluted for antibody production in mice. Processing of the VP-polyprotein by 3C protease was carried out in the in vitro co-transcription/translation system and detected by Western blot. The results showed there were protein bands corresponding to the sizes of VP2+VP1 and VP1+VP3 indicating that the processing might have been partial or incomplete.

Hemophilus influenzae (Hi) has been an important strain in clinical examination, but it is not clear about its subtype, non-typable Hi, in causing invasive diseases after years of application of vaccines against typable Hi. Thus, the study is to determine the major infected bacterium causing invasive diseases and investigate the genotype relationship between antibiotic resistance and active IgA1 protease. Practical approaches of the study include clone each microorganism from infected blood, pus, sputum, bronchial washing and thorax samples of patients with invasive diseases. Each of the organisms was assayed for IgA1 protease activity, the type of the enzyme and antibiotic resistance. Forty-five patients aged 1 to over 71 with invasive diseases of diagnosed pneumonia, sinusitis, bacteremia, bronchitis, chronic obstructive of pulmonary diseases (COPD), conjunctivitis or otitis media, were analyzed, and all the 45 Hi isolates contain iga gene but only 80% contain active IgA1 protease. Mutations to silence iga gene are common in Hi isolates. The dominant population of infected bacterium is Hi, 84% of which are non-typable (NTHi). About 76% of NTHi and 85% of typable Hi (THi) contained active IgA1 protease. PFGE analysis showed that none of the 45 Hi isolates had identical genome. Phenotypes of active IgA1 protease and antibiotic resistance of the 45 Hi isolates showed no close relations each other. This study clearly demonstrated that NTHi has become a dominant strain in causing invasive diseases. Antibiotic resistance and active IgA1 protease are two essential but independent phenotypes for NTHi to infect and colonize. Antibiotic resistance of NTHi is dependent on the presence of beta-lactamase.

Synthetic biology is a nascent discipline that aims to design and construct new biological systems beyond those found in nature, ultimately using them to probe, control, or even replace existing biological systems. The success of synthetic biology depends on the assembly of a set of well-defined and modular tools. These tools should ideally be mutually compatible, reusable in different contexts, and have minimum crosstalk with endogenous proteins of the subject. The tobacco etch virus protease (TEV protease, TEVp) is a suitable candidate for such a tool due to its unique substrate specificity and high efficiency. Importantly, TEVp is capable of imitating proteolysis, a ubiquitous mechanism in nature for post-translational modifications and signal propagation. Here, TEVp is employed as a self-contained proteolytic device capable of executing biological tasks that are otherwise governed by endogenous proteins and processes. Consequently, the goal of using TEVp for synthetic manipulation of biosystems is achieved. First, a single-vector multiple gene expression strategy utilizing TEVp self-cleavage was created. This approach was used for the robust expression of up to three genes in both bacterial and mammalian cells with consistent stoichiometry. The products can then be individually purified or targeted to distinct subcellular compartments respectively. Second, a temperature-inducible TEVp was created by incremental truncation of TEVp. The 18th truncation of TEVp (tsTEVp) resulted in negligible activity at 37 °C, but retained sufficient activity at 30 °C for rapid processing of its substrates in several mammalian cell cultures. Finally, tsTEVp was applied in the context of other synthetic modules to generate a variety of biological responses. Its versatility was demonstrated as cellular processes including protein localization, cellular blebbing, protein degradation, and cell death were rewired to respond to the physical stimulus of temperature.

Synthetic biology is a nascent discipline that aims to design and construct new biological systems beyond those found in nature, ultimately using them to probe, control, or even replace existing biological systems. The success of synthetic biology depends on the assembly of a set of well-defined and modular tools. These tools should ideally be mutually compatible, reusable in different contexts, and have minimum crosstalk with endogenous proteins of the subject. The tobacco etch virus protease (TEV protease, TEVp) is a suitable candidate for such a tool due to its unique substrate specificity and high efficiency. Importantly, TEVp is capable of imitating proteolysis, a ubiquitous mechanism in nature for post-translational modifications and signal propagation. Here, TEVp is employed as a self-contained proteolytic device capable of executing biological tasks that are otherwise governed by endogenous proteins and processes. Consequently, the goal of using TEVp for synthetic manipulation of biosystems is achieved. First, a single-vector multiple gene expression strategy utilizing TEVp self-cleavage was created. This approach was used for the robust expression of up to three genes in both bacterial and mammalian cells with consistent stoichiometry. The products can then be individually purified or targeted to distinct subcellular compartments respectively. Second, a temperature-inducible TEVp was created by incremental truncation of TEVp. The 18th truncation of TEVp (tsTEVp) resulted in negligible activity at 37 °C, but retained sufficient activity at 30 °C for rapid processing of its substrates in several mammalian cell cultures. Finally, tsTEVp was applied in the context of other synthetic modules to generate a variety of biological responses. Its versatility was demonstrated as cellular processes including protein localization, cellular blebbing, protein degradation, and cell death were rewired to respond to the physical stimulus of temperature.

No description available.

Diss. (sammanfattning) Uppsala : Univ., 2001. / Härtill 4 uppsatser.