Doctor of Philosophy
Chemical and Biochemical Engineering
Prof. A. Bassi
Prof. J. Zhu
Biotechnology has significantly contributed to the production of food, drugs, and a lot of specialty and chemical products. In addition to that, biotechnology provided and can still provide sustainable, and green, solutions to many environmental problems. By using recombinant DNA technology, almost any protein can be cloned and produced in large quantities. The major challenge facing this type of a protein production approach is the need for proper refolding of target protein. The main objective of the current study is to provide the biotechnology industries with a more efficient and economical technology for continuous protein purification and refolding. The Circulating fluidized bed technology has been selected as a promising alternative to the current practice in protein purification and refolding. The circulating fluidized bed technology offers many advantages over conventional technique such as better heat and mass transfer, better solid liquid contact, ability to operate in continuous mode, the ability to handle two main liquid streams without mixing.
During the course of this study matrix-assisted protein refolding using Immobilized Metal affinity chromatography (IMAC) was successfully used for purification and refolding of recombinant GST-His6 protein. The recombinant protein was produced in genetically modified E. coli and the over expression of target protein was induced by IPTG.
In addition, two high density composite IMAC beads have been successfully designed and constructed. The main components in IMAC beads are biopolymer agarose and a filler. The biopolymer outer layer was derivatized to introduce the IMAC chelating moiety IDA. The constructed composite IMAC beads were fully characterized by SEM, FTIR, Master Sizer, and XPS to confirm the chemical structure of the derivatized biopolymer layer on the beads surface. Other commercially available IMAC beads were also evaluate during the course of this study.
A new scale-down design of the Liquid solid circulated fluidized bed system was constructed to investigate the developed protein purification and refolding approach in a continuous operation mode. The new LSCFB system has a simple design with some unique features. The total volume of the new LSCFB system is about 2.85 L and can handle up to 1 kg of Ion-exchange beads in a continuous mode. The system was successfully used for purification and refolding of His6-tagged recombinant proteins. The new system can also be applied for purification of many other biological products. The newly designed LSCFB system has high potential in environmental applications.
Alassuity, Ahmed, "Continuous protein refolding in a liquid solid circulating fluidized bed (LSCFB) system" (2017). Electronic Thesis and Dissertation Repository. 4756.
Available for download on Tuesday, December 31, 2030