Production of biologically active recombinant HIV-1 protease and intehrase for the purpose of screening medicianl plant extracts
- Authors: Bosch, Janine
- Date: 2009
- Subjects: Medicinal plants -- South Africa , HIV infections -- Alternative treatment -- South Africa , Materia medica, Vegetable -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10325 , http://hdl.handle.net/10948/1056 , Medicinal plants -- South Africa , HIV infections -- Alternative treatment -- South Africa , Materia medica, Vegetable -- South Africa
- Description: Human immunodeficiency virus (HIV) and its gradual weakening of the immune system is an ever growing threat. Acquired immune deficiency syndrome (AIDS), the final stage of HIV, renders a person vulnerable to various opportunistic infections, which in the end lead to death. Apart from intensive vaccine studies, treatment research mainly focuses on preventing the individual HIV enzymes (reverse transcriptase, integrase and protease) from performing their functions. Entry inhibitors, however, block viral entry into the cell, while antisense drugs lock onto the viral genome to keep it from functioning. In this study production of active recombinant HIV-1 protease and integrase was attempted for future drug screening programs. HIV-1 protease was cloned into a pET28b(+) vector and expressed in ROSETTA(DE3)pLysS cells. The protein was purified using a nickel-affinity column utilizing the hexa-histidine tag encoded by the vector. Gel filtration chromatography was attempted after refolding of the protease, but protease yield seemed to decrease with the additional purification step. Partially purified protease was characterized with kinetic studies. Kinetic parameters of HIV-1 protease were determined to be Km = 592 μM, Vmax = 0.59 μM/min and kcat = 31 s-1. HIV-1 integrase, which was cloned into a pET15b vector, was expressed in E. coli BL21(DE3) cells. The coding sequence had been mutated to introduce the amino acid substitutions F185K and C280S, increasing solubility of the protein. The first step in purification of this protein was nickel-affinity chromatography, after which cation exchange chromatography was attempted. HIV-1 integrase concentration was low throughout experiments and no clear elution from the cation exchange column could be observed. A non-radioactive enzyme linked HIV-1 integrase assay failed to detect integrase activity. Modifications to future studies of the integrase are suggested in the chapter involved.
- Full Text:
- Date Issued: 2009
- Authors: Bosch, Janine
- Date: 2009
- Subjects: Medicinal plants -- South Africa , HIV infections -- Alternative treatment -- South Africa , Materia medica, Vegetable -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10325 , http://hdl.handle.net/10948/1056 , Medicinal plants -- South Africa , HIV infections -- Alternative treatment -- South Africa , Materia medica, Vegetable -- South Africa
- Description: Human immunodeficiency virus (HIV) and its gradual weakening of the immune system is an ever growing threat. Acquired immune deficiency syndrome (AIDS), the final stage of HIV, renders a person vulnerable to various opportunistic infections, which in the end lead to death. Apart from intensive vaccine studies, treatment research mainly focuses on preventing the individual HIV enzymes (reverse transcriptase, integrase and protease) from performing their functions. Entry inhibitors, however, block viral entry into the cell, while antisense drugs lock onto the viral genome to keep it from functioning. In this study production of active recombinant HIV-1 protease and integrase was attempted for future drug screening programs. HIV-1 protease was cloned into a pET28b(+) vector and expressed in ROSETTA(DE3)pLysS cells. The protein was purified using a nickel-affinity column utilizing the hexa-histidine tag encoded by the vector. Gel filtration chromatography was attempted after refolding of the protease, but protease yield seemed to decrease with the additional purification step. Partially purified protease was characterized with kinetic studies. Kinetic parameters of HIV-1 protease were determined to be Km = 592 μM, Vmax = 0.59 μM/min and kcat = 31 s-1. HIV-1 integrase, which was cloned into a pET15b vector, was expressed in E. coli BL21(DE3) cells. The coding sequence had been mutated to introduce the amino acid substitutions F185K and C280S, increasing solubility of the protein. The first step in purification of this protein was nickel-affinity chromatography, after which cation exchange chromatography was attempted. HIV-1 integrase concentration was low throughout experiments and no clear elution from the cation exchange column could be observed. A non-radioactive enzyme linked HIV-1 integrase assay failed to detect integrase activity. Modifications to future studies of the integrase are suggested in the chapter involved.
- Full Text:
- Date Issued: 2009
In vitro testing to investigate the anticoagulant/antithrombotic and antidiabetic biological activity of Leonotis Leonurus
- Authors: Mnonopi, Nandipha Olivia
- Date: 2007
- Subjects: Leonotis leonurus -- Physiological aspects , Medicinal plants -- South Africa , Materia medica, Vegetable -- South Africa , Plant bioactive compounds
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10331 , http://hdl.handle.net/10948/693 , Leonotis leonurus -- Physiological aspects , Medicinal plants -- South Africa , Materia medica, Vegetable -- South Africa , Plant bioactive compounds
- Description: The rising costs of prescription drugs in the maintenance of personal health and wellbeing have increased the interest in medicinal plants. The World Health Organization estimates that 65 percent-80 percent of the world’s population use traditional medicine as their primary form of health care. In this project the focus has been on the use of Leonotis leonurus extracts as a traditional medicine. The major chemical constituent of this plant is marrubiin, which is a diterpenoid labdane lactone formed from a precursor called premarrubiin. Aqueous and acetone extract (AL and OL extract, respectively) of this plant has been found to have an antithrombotic effect, with IC50 values of 3mg/ml and 6mg/ml, respectively. The extracts also have an effect on fibrinolysis, where the lysis time was decreased by more than 50 percent by the organic extract and standard marrubiin. In whole blood ADP-induced platelet aggregation, the organic extract inhibited aggregation by 68 percent at a final concentration of 138μg/ml (equivalent to 7.2μg/ml marrubiin). Marrubiin has also been screened for antithrombotic/anticoagulant activity; no antithrombotic activity has been observed but it increased the rate of fibrinolysis, by decreasing lysis time by 64 percent and also decreasing fibrin formation. From these findings it can be concluded that marrubiin has a fibrinolytic effect and antiplatelet aggregation effect. In the diabetic studies, in hyperglycemic condition, the OL (10μg/ml) extract and standard marrubiin significantly increased insulin secretion by 200 percent (2-fold) and 400 percent (4-fold), respectively, with respect to the control. The OL extract and standard marrubiin stimulated the release of insulin, the stimulatory index was significantly increased by 450 percent (4.5-fold) and 500 percent (5-fold), respectively, with respect to the control. In the apoptotic studies, in the normoglycemic and hyperglycemic conditions, the OL extract decreased the occurrence of apoptosis, in a dose-dependent manner, with the lower concentrations inducing apoptosis significantly higher than the relevant controls. Standard marrubiin did not have an effect on apoptosis in hyperglycemic condition, but it decreased the occurrence of apoptosis by 200 percent (2-fold) under normoglycemic conditions. The OL extract increased proliferation by 148 percent (1.48- fold) and 155 percent (1.55-fold) in normoglycemic and hyperglycemic conditions, respectively. The same effect was observed for standard marrubiin, where, proliferation was increased by 180 percent (1.8-fold) and 200 percent (2.0-fold) in normoglycemic and hyperglycemic conditions, respectively. RT-PCR displayed that standard marrubiin inhibited the expression of insulin by 50 percent under normoglycemic conditions.
- Full Text:
- Date Issued: 2007
- Authors: Mnonopi, Nandipha Olivia
- Date: 2007
- Subjects: Leonotis leonurus -- Physiological aspects , Medicinal plants -- South Africa , Materia medica, Vegetable -- South Africa , Plant bioactive compounds
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10331 , http://hdl.handle.net/10948/693 , Leonotis leonurus -- Physiological aspects , Medicinal plants -- South Africa , Materia medica, Vegetable -- South Africa , Plant bioactive compounds
- Description: The rising costs of prescription drugs in the maintenance of personal health and wellbeing have increased the interest in medicinal plants. The World Health Organization estimates that 65 percent-80 percent of the world’s population use traditional medicine as their primary form of health care. In this project the focus has been on the use of Leonotis leonurus extracts as a traditional medicine. The major chemical constituent of this plant is marrubiin, which is a diterpenoid labdane lactone formed from a precursor called premarrubiin. Aqueous and acetone extract (AL and OL extract, respectively) of this plant has been found to have an antithrombotic effect, with IC50 values of 3mg/ml and 6mg/ml, respectively. The extracts also have an effect on fibrinolysis, where the lysis time was decreased by more than 50 percent by the organic extract and standard marrubiin. In whole blood ADP-induced platelet aggregation, the organic extract inhibited aggregation by 68 percent at a final concentration of 138μg/ml (equivalent to 7.2μg/ml marrubiin). Marrubiin has also been screened for antithrombotic/anticoagulant activity; no antithrombotic activity has been observed but it increased the rate of fibrinolysis, by decreasing lysis time by 64 percent and also decreasing fibrin formation. From these findings it can be concluded that marrubiin has a fibrinolytic effect and antiplatelet aggregation effect. In the diabetic studies, in hyperglycemic condition, the OL (10μg/ml) extract and standard marrubiin significantly increased insulin secretion by 200 percent (2-fold) and 400 percent (4-fold), respectively, with respect to the control. The OL extract and standard marrubiin stimulated the release of insulin, the stimulatory index was significantly increased by 450 percent (4.5-fold) and 500 percent (5-fold), respectively, with respect to the control. In the apoptotic studies, in the normoglycemic and hyperglycemic conditions, the OL extract decreased the occurrence of apoptosis, in a dose-dependent manner, with the lower concentrations inducing apoptosis significantly higher than the relevant controls. Standard marrubiin did not have an effect on apoptosis in hyperglycemic condition, but it decreased the occurrence of apoptosis by 200 percent (2-fold) under normoglycemic conditions. The OL extract increased proliferation by 148 percent (1.48- fold) and 155 percent (1.55-fold) in normoglycemic and hyperglycemic conditions, respectively. The same effect was observed for standard marrubiin, where, proliferation was increased by 180 percent (1.8-fold) and 200 percent (2.0-fold) in normoglycemic and hyperglycemic conditions, respectively. RT-PCR displayed that standard marrubiin inhibited the expression of insulin by 50 percent under normoglycemic conditions.
- Full Text:
- Date Issued: 2007
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