Advancing Rhodobacter sphaeroides for Functional Expression of Medically and Industrially Relevant Membrane Proteins

Open Access
Author:
Erbakan, Mustafa
Graduate Program:
Bioengineering
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
June 19, 2014
Committee Members:
  • Wayne Roger Curtis, Dissertation Advisor
  • Wayne Roger Curtis, Committee Chair
  • Peter J Butler, Committee Member
  • Sheereen Majdzarringhalamaraghy, Committee Member
  • Manish Kumar, Committee Member
Keywords:
  • Rhodobacter sphaeroides
  • Membrane Proteins
  • Heterologous Expression
  • Aquaporin
  • Cellulose synthase
  • Proteopolymersome
Abstract:
Membrane proteins have important roles in physiology of biological cells and they comprise the largest group of drug targets making them an attractive field of reasearch. Availability of membrane proteins is limited due to their low abundance in natural sources and low expression titers in heterologous expression systems. In the work presented; we employed Rhodobacter sphaeroides 2.4.1., a purple photosynthetic bacterium, for expression of functional membrane proteins with medical and industrial relevance. The breadth of the expression system was tested with several membrane proteins from distinct families including Cytochrome-cy from Rhodobacter capsulatus; GhoT from Escherichia coli; human Occludin; human AQP9, a human water channel protein; BcsAB complex from Rhodobacter sphaeroides and finally R. sphaeroides RsAqpZ. Target proteins were overexpressed, purified and their functionality was determined by specific activity assays. Expression titers were significantly improved for AQP9, BcsAB and Occludin compared to previous studies. RsAqpZ was overexpressed at ~20 mg/L, characterized and evaluated for utilization in biomimetic water desalination membranes made from tri-block copolymers. RsAqpZ proteopolymersomes had superior water permeability compared to the reports for E. coli AqpZ, which is commonly used in aquaporin based water desalination membranes. A new method based on fluorescence correlation spectroscopy (FCS) was developed to determine single channel properties of membrane proteins. FCS was used to determine the number of membrane protein per lipid vesicle, which in combination with stopped flow light scattering for permeability measurement of vesicles, enabled calculation of single channel water permeability. It also enabled determination of reconstitution efficiency of RsAqpZ into phospholipid vesicles. C-terminal mBanana fluorescent protein fusion of RsAqpZ was used as the fluorescent tag. The presence of mBanana did not affect the water permeability as measured by stopped-flow light scattering. Thus, single channel permeability and insertion efficiency of RsAqpZ was correctly determined by combining the FCS and stopped-flow light scattering experiments. Finally, a thin film trickle-bed photobioreactor prototype for high density Rhodobacter culturing was built and initial characterization was carried out, in which light penetration problems were addressed.