Development Of Optimization Procedure For The Expression Of The Membrane Protein Aquaporin In Rhodobacter Sphaeroides

Open Access
Author:
Escotet Espinoza, Manuel Sebastian
Area of Honors:
Chemical Engineering
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Wayne Roger Curtis, Thesis Supervisor
  • Darrell Velegol, Honors Advisor
  • Manish Kumar, Faculty Reader
Keywords:
  • Aquaporin
  • membrane protein
  • fluorescence
  • genetic engineering
  • screening
  • western blot
Abstract:
Membrane proteins are the new targets for many researchers in the field of biotechnology and medicine. One of the problems regarding their study is low abundance in vivo and limited availability of functional protein for in vitro studies. Rhodobacter sphaeroides is being developed as a highly effective production organism for the expression of biotechnologically relevant membrane proteins. R. sphaeroides is being used developed as a membrane protein platform due to the organism’s large intracellular membrane level and potential for high biomass yields (>4 gDW/L) in scalable anaerobic photoheterotrophic systems. The production of the water-transport membrane protein Aquaporin was studied, quantified and analyzed through the use fluorescent fusion and poly-histidine tags. Correlations between culture fluorescence and Western blot densitometry were validated as a simple screening method for rapidly predicting and quantifying the membrane protein levels. The values for fluorescence and Western Blotting were correlated to each other and shown to correlate with standard pure protein values. Fluorescence per optical density (OD) therefore provides a rapid measurement of membrane protein expression per cell. To demonstrate the utility of this correlation for optimizing the protein expression, temperature effects were studied as a simple control parameter in our system. Room and optimal temperatures conditions (25°C and 32°C, respectively) for Rhodobacter sphaeroides were studied under the anaerobic photoheterotrophic conditions required for protein expression. The results from the experiments indicated that the production of protein per cell showed similar trend at both temperatures, though the total productivity was much higher at 32°C due to faster growth. This work also identifies several opportunities for improving this screening method for membrane expression. Although the fluorescent fusion proteins served as a simple and effective method for determining the protein concentration in vivo, the use of obligate aerobic fluorescent proteins presented a significant experimental inconvenience for the measurement of the fluorescent readings due to the use of the anaerobic conditions. The maturation (folding and optimal fluorescence) requires oxygen exposure after sampling. The observed maturation time of more than 40 hours undermined the goal of rapid screening. Future research will seek to overcome this limitation by using a new generation of facultative anaerobic fluorescent proteins such as riboflavin-binding proteins.