Date of Award
Master of Science In Chemical Engineering Degree
Chemical And Biomedical Engineering
Dr. Marvin Thrash
Dr. Christopher Wirth
The ability to express and purify large quantities of recombinant protein allows for biotechnological applications such as: protein characterization, usage in industrial processes, the development of commercial goods, and other advanced studies. In the classical methods of protein expression, cells are grown to the mid-log phase and are induced via a simple sugar or a chemical agent. Post-induction, the energy used for cellular growth is redirected for the production of ELPs. Samples are collected and analyzed for cell count and harvested for fluorescence spectroscopy and protein purification. Optimization of protein expression of an elastin-like-polypeptide and green fluorescence protein fusion (ELP-GFP fusion protein) is achieved by targeting the organisms, expression systems, altering media formulations, antibiotic resistance, and selective inducers for optimal production of ELP-GFP fusion proteins. We investigate which organism and expression system, after optimizing the conditions, yields maximum amount of ELP-GFP fusion protein by quantifying with fluorescence and absorbance at 280 nm. The bacterial host, BL21 (DE3), gave optimal expression of ELP-GFP when induced with lactose and galactose. The yields were between 400-600 mg/L of culture, much higher than when expressed in LB media and induced with IPTG. The Vmax strain, which has a doubling rate of less than 10 minutes, however, it is not optimized for protein expression. For a different ELP-GFP fusion protein, the protein yields after purification v were between 200-300 mg/L for BL21 (DE3) and between 100-200 mg/L for Vmax derived cells. The lac and the ara operons do not behave the same way during protein expression. The lac system suffers from “leaky” expression whereas the ara system is more tightly regulated. Fluorescence was used as a reliable means to understand the behavior of my ELP-GFP proteins. The fluorescence data revealed that the lac operon is more stable for longer fermentations times whereas, the ara operon is more suitable for shorter fermentation times.
Quiroga iii, Blas, "Optimizing Protein Expression of An Elastin Like Polypeptidegreen Fluorescent Protein Fusion In Bacterial Systems" (2021). ETD Archive. 1228.