A Low-Cost Kit for the Purification of a Mannan-Binding Lectin Preventing HIV Transmission
Open Access
- Author:
- Krum, David
- Area of Honors:
- Chemical Engineering
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Wayne Roger Curtis, Thesis Supervisor
Wayne Roger Curtis, Thesis Honors Advisor
Amir Sheikhi, Faculty Reader - Keywords:
- HIV
cyanobacteria
chemical engineering
protein purification
PCR
Modeling
Transgenic Plants
Microscopy - Abstract:
- Human Immunodeficiency Virus (HIV) is a devastating, sexually transmitted virus that is particularly prevalent in Sub Saharan Africa. The lack of access to anti-retroviral therapy in this region, which prevents an infected individual from spreading the virus to others, means that the virus is transmitted easily. Mannose-binding lectins, a type of carbohydrate-binding protein, from cyanobacteria have been shown to selectively bind to viral envelope glycoproteins. These lectins prevent interaction of HIV particles with the CD4 receptor on T-cells, preventing a transmission event. This work sought to develop a cheap, easy-to-use protein production platform in photosynthetic cyanobacteria for a lectin protein that prevents transmission of HIV. Microvirin, a mannose-binding lectin found in Microcystis aeruginosa, was fused to an elastin-like-polypeptide inverse solubility aggregation tag, with an interceding pH-inducible intein. Synechococcus elongatus was selected as a production platform, as it is phototrophic and can be easily genetically transformed to produce recombinant proteins. The fusion protein was inserted into the obligatory cyanobacteria plasmid pAQ1 for transformation into Synechococcus elongatus. This system allows for the selective purification of microvirin from a cyanobacterial lysate based on temperature-mediated solubility changes and pH-mediated intein cleavage. This system is designed to not require antibiotic selection pressure, and is fully phototrophic, meaning that the costs of maintaining the bacterial culture are very low. The cloning associated with this project proved to be very difficult, and ligating the microvirin fusion protein into the pAQ1 plasmid limited the progress of the project. The cloning efforts undertaken are detailed in this thesis, and the current state of the project is described. Furthermore, future work and experiments that will need to be conducted to characterize the purification system are described in detail.