Calcineurin (CaN), a member of ser/thr protein phosphatase, was cloned from bovine retina. The peptide sequence of CaN A subunit is consisted of 511 amino acid residues. A 10 amino acid (A-T-V-E-A-I-E-A-D-E-A) deletion before the autoinhibitory domain was observed in bovine retina CaN A compared to bovine brain CaN A. The study on CaN activity and regulation demonstrated that different metal ions have different effects on its phosphatase activity. Ni2+ was found to be the strongest stimulator while Zn2+ was found to inhibit CaN phosphatase activity. Mn2+ was a relatively less effective stimulator compared to Ni2+. Fe2+ was also able to stimulate CaN phosphatase activity; in contrast, a previous study found Fe2+ slightly inhibited bovine brain CaN activity. The residues at 97-201 were found to be essential for bovine retina CaN A phosphatase activity. The residues at 407-456 also had an inhibitory effect on CaN A phosphatase activity in addition to the previously known auto inhibitory domain at 457-480. These observations suggest that bovine retina CaN A might possess some distinct structural characteristics compared to bovine brain CaN A.

Calcineurin (CaN), a member of ser/thr protein phosphatase, was cloned from bovine retina. The peptide sequence of CaN A subunit is consisted of 511 amino acid residues. A 10 amino acid (A-T-V-E-A-I-E-A-D-E-A) deletion before the autoinhibitory domain was observed in bovine retina CaN A compared to bovine brain CaN A. The study on CaN activity and regulation demonstrated that different metal ions have different effects on its phosphatase activity. Ni2+ was found to be the strongest stimulator while Zn2+ was found to inhibit CaN phosphatase activity. Mn2+ was a relatively less effective stimulator compared to Ni2+. Fe2+ was also able to stimulate CaN phosphatase activity; in contrast, a previous study found Fe2+ slightly inhibited bovine brain CaN activity. The residues at 97-201 were found to be essential for bovine retina CaN A phosphatase activity. The residues at 407-456 also had an inhibitory effect on CaN A phosphatase activity in addition to the previously known auto inhibitory domain at 457-480. These observations suggest that bovine retina CaN A might possess some distinct structural characteristics compared to bovine brain CaN A.

The addition of Mn(II) to an early stationary-phase Deinococcus radiodurans RI culture could induce a new round of cell division (MnCD effect). The addition of Mn(II) could also stimulate the utilization of glucose and fructose in this bacterium. Class II fructose-1,6-bisphosphate aldolase (FBA) is an Mn-dependent key enzyme in pentose phosphate pathway. Therefore, in this research, we focused on the studies of the fba gene. Base on the gene sequence, FBA protein was composed of 306 amino acids, (M.W., 32.4 kDa¡F pI, 5.4). The expected PCR product size of the fba gene is 9.3 kbp. We had amplified the fba gene by using both Taq DNA polymerase and pfu turbo DNA polymerase. The sequence of the pfu turbo DNA polymerase products showed a higher homology with the fba gene than those of using Taq DNA polymerase. These amplified fba gene was cloned into three expression vectors, pGEX-4T-2, pQE30, and pET28a, and then further expressed in E. coli BL21(DE3)RIL and JM109. The recombinant GST-FBA protein could be overproduced in pTDA2/BL21(DE3)RIL. However, the expressed insoluble protein accumulated as inclusion bodies in the cells and exhibited no enzyme activity. After partial purification, and processing by thrombin protease cleavage, urea treatment, and the addition of Mn(II), this enzyme still showed no activity. The recombinant pEDA2/BL21(DE3)RIL strain cells grew in 18¢J and induced by 0.1mM IPTG could produced a soluble form His-Thrombin-T7-FBA protein which performed a 50X higher activities than those cells grew in 30¢J. This result indicated that decreasing the indicatioin temperature could improve the protein solubility and activity.

Thesis (Ph. D.)--University of Texas at Austin, 1999. / Vita. Greek alphabet delta in title. Includes bibliographical references (leaves 136-155). Available also in a digital version from Dissertation Abstracts.

Thesis (Ph. D. in Microbiology)--Washington State University. / Includes bibliographical references.

Thesis (M. Phil.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 74-85).

Thesis (Ph. D.)--University of Missouri-Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves [100]-114). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves [100]-114). Also available on the Internet.

Burkholderia pseudomallei and Burkholderia mallei cause the diseases melioidosis and glanders, respectively. These diseases are endemic mainly in southeastern Asia and northern Australia, but they also pose a bioterrorism threat in the developed world. These diseases have high mortality, partially due to the lack of vaccines and rapid, accurate diagnostic assays. The work discussed here represents a part of a larger project to develop a dependable diagnostic assay for use in both developing endemic areas and the developed world, as well as a subunit vaccine to protect against disease. In this study, several proteins from B. pseudomallei, B. mallei, and the closely related but less virulent B. thailandensis have been cloned, expressed and purified in order to develop highly sensitive and specific diagnostic reagents for the detection of B. pseudomallei and B. mallei in infected patient samples. Protein targets expressed in this study were also used in subunit vaccine development for melioidosis and glanders. / text

published_or_final_version / abstract / toc / Zoology / Doctoral / Doctor of Philosophy