Date of Graduation
Bachelor of Science in Chemical Engineering
Beitle , Robert
Annexin-V is a cytosolic protein involved in many significant physiological roles including stabilization of the phospholipid membrane, regulation of blood coagulation, and cell apoptosis or programmed cell death. Though the exact mechanism of Annexin-V in the human body is not fully understood, its wide range of applications in the medical field cannot be denied. Annexin-V can be applied in cancer therapy as a diagnostic to detect cell apoptosis and determine the effectiveness of chemotherapeutic drugs. Furthermore, Annexin-V could even be used as a drug delivery vehicle in the treatment of cancer. Commercially useful proteins like Annexin-V are in high demand, however, one big obstacle is that yielding a large amount of pure Annexin-V could be a tedious and expensive procedure. Performing research with this protein could also become a huge financial undertaking. The focus of this project was to study the optimal conditions by which the Annexin-V protein could be expressed, purified, and mass-produced. An additional goal was to devise a cost-effective procedure of mass-producing Annexin-V. SDS-PAGE was the primary method applied to test the intensity and purity of the Annexin-V sample yielded from experiments. SDS-PAGE results establish that the E. coli over-expressed the Annexin-V protein. Purification using an ion exchange (DEAE) column yielded Annexin-V of relatively high purity; a step-wise salt gradient was significantly more effective than a linear salt gradient in eluting a more pure product from the column. A bioreactor was used to perform batch fermentation. Three types of media were used for the batch experiments: LB broth only, LB and 9 g/L glucose, and M9+glucose media consisting of M9 salts, trace elements, biotin, and 9 g/L glucose. Among the batch fermentations, LB and 9 g/L glucose yielded the highest amount of Annexin-V. However, the E. coli were not able to successfully grow in the M9 media mixture. Additional experiments using the M9 media will have to be performed to verify this observation. The data collected throughout this project suggest that: 1) E. coli cultures are effective in over-expressing Annexin-V, 2) A DEAE purification column, accompanied by the use of a step-wise salt gradient, yields Annexin-V of considerably high purity 3) The use of LB/glucose media is an effective method to mass-produce Annexin-V.
Suresh Kumar, G. (2014). Expression, purification, and large-scale production of the human recombinant Annexin-V protein. Chemical Engineering Undergraduate Honors Theses Retrieved from https://scholarworks.uark.edu/cheguht/46