Immunoglobulin A (IgA), a major serum immunoglobulin and a predominant antibody in the external secretions that bathe mucosal surfaces, plays key roles in immune protection. Some pathogenic bacteria including Haemophilus influenzae and Neisseria meningitides, however, produce a protease called IgA1 protease to impair the function of IgA1. The iga mRNA is initially translated into a large precursor containing four distinct domains: a 31-amino acid signal peptide which leads the precursor to the periplasmic space, an 105-kDa protease domain which cleaves host IgA1 molecule, a £]-domain responsible for autotransportation of the protease domain, and a linker £\-protein between the protease and the £]-domain. The hydrolytic function of the protease and the role of the £]-core had been studied extensively, but the role of the £\-protein has never been studied. Thus this study is designed to reveal the possible functions of £\-protein in the proliferation of lymphocytes. To complete the project, PCR was used to amplify the DNA fragment for £\-protein using iga gene (Gene Bank DQ683357) as template. The fragment spans nucleotide numbers of 1015-1405. The fragment was then cloned into pGEX-2T for expression. Recombinant £\-protein was purified using glutathione -Sepharose column. The purified recombinant protein did not seem to affect the cell growth at the concentration of 1 £gg/ml compared with the medium control or GST control. Interestingly, when the concentration was increased to 5 £gg/ml or 10 £gg/ml, £\-protein seems to enhance the cell growth on the 2nd, 4th and 6th day assays, The results suggested that £\-protein may enhance the cell growth in 4 days.

No description available.

published_or_final_version / abstract / Zoology / Doctoral / Doctor of Philosophy

No description available.

We have formed the basis for a synthetic gene regulatory network in S. cerevisiae to assess the relative contribution of proteins and protein interactions to the function of transcriptional regulatory complexes. This gene network contains two synthetic transcriptional regulator (TR) fusion proteins that control transcriptional activation and repression of a reporter expressed by a synthetic promoter. This system can be modulated and quantified specifically by our manipulations since the transcriptional regulators (TRs) are only active when drug induced. In this thesis, I characterized and optimized my gene network to enable accurate measurements of transcriptional regulation. I demonstrated that my synthetic repressor interacts with a selected native transcriptional regulatory complex. Future mutation experiments can be performed to investigate the relative importance that the selected protein interaction plays on transcriptional regulation. New synthetic repressor fusion proteins can be created to study the relative importance of protein interactions between other TRs, making this tool versatile. This tool will allow us to quantify interactions and gain a deeper understanding of the complicated mechanisms controlling transcriptional regulation.

No description available.

No description available.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

It is critical to consider the balance between the catalytic capabilities of an enzyme and the inherent structural stability of the protein when developing enzymes for specific applications. Rational site directed mutagenesis has been used to explore the role of residues 254 and 257 in the global stability and catalytic specificities of organophosphorus hydrolase (OPH, EC 3.1.8.1). Substitution of residues H254 and H257, which are located near the active site, had a marked effect on both the global stability and substrate specificity of the enzyme. For example, the for the double mutation CoTGΔ2+ H254R H257L (RL) enzyme variant was 19.6 kcal/mol, 5.7 kcal/mol less than that of the wild type enzyme. At the same time, the altered enzyme was catalytically more effective against VX and VR (Russian VX), as compared to the wild type enzyme. Limited proteolysis verified the importance of residues 254 and 257 for functional stability, evidenced by enhanced resistance to irreversible unfolding associated with thermal denaturation. It has been possible to construct third generation OPH variants, which are more stable than the wild type enzyme, with a 10 °C increase in the apparent melting temperature (TM app), yet retained desirable catalytic properties. It appeared that aromatic stacking and cation-π interactions involving near active site residues not only affected activity but significantly contributed to the chemical and thermal stability of OPH. Rational design was used to develop an enzyme with an optimized orientation on a catalytically active biosensor surface. In these studies, lysine side chains located on the surface of OPH were used to create attachment sites to a surface plasmon resonance sensor resulting in an ensemble of enzyme orientations. Some of these orientations could be functionally restrictive if the active site is oriented toward the sensor surface. Substitution of a lysine near the active site resulted in 20% more activity with 53% less enzyme immobilized, thus increasing the specific activity of the decorated surface 2.5 fold.

It is critical to consider the balance between the catalytic capabilities of an enzyme and the inherent structural stability of the protein when developing enzymes for specific applications. Rational site directed mutagenesis has been used to explore the role of residues 254 and 257 in the global stability and catalytic specificities of organophosphorus hydrolase (OPH, EC 3.1.8.1). Substitution of residues H254 and H257, which are located near the active site, had a marked effect on both the global stability and substrate specificity of the enzyme. For example, the for the double mutation CoTGΔ2+ H254R H257L (RL) enzyme variant was 19.6 kcal/mol, 5.7 kcal/mol less than that of the wild type enzyme. At the same time, the altered enzyme was catalytically more effective against VX and VR (Russian VX), as compared to the wild type enzyme. Limited proteolysis verified the importance of residues 254 and 257 for functional stability, evidenced by enhanced resistance to irreversible unfolding associated with thermal denaturation. It has been possible to construct third generation OPH variants, which are more stable than the wild type enzyme, with a 10 °C increase in the apparent melting temperature (TM app), yet retained desirable catalytic properties. It appeared that aromatic stacking and cation-π interactions involving near active site residues not only affected activity but significantly contributed to the chemical and thermal stability of OPH. Rational design was used to develop an enzyme with an optimized orientation on a catalytically active biosensor surface. In these studies, lysine side chains located on the surface of OPH were used to create attachment sites to a surface plasmon resonance sensor resulting in an ensemble of enzyme orientations. Some of these orientations could be functionally restrictive if the active site is oriented toward the sensor surface. Substitution of a lysine near the active site resulted in 20% more activity with 53% less enzyme immobilized, thus increasing the specific activity of the decorated surface 2.5 fold.