(294f) Use of Fluorescent Protein Fusions to Optimize Membrane Protein Expression in Anaerobic Photoheterotrophic Rhodobacter

We describe the use of fluorescent protein fusions as a method for optimizing and accurately quantifying membrane protein expression in Rhodobacter.  Membrane proteins (MP) are a difficult class of proteins to study; however, their medical and biotechnical importance makes them compelling to address the associated challenges. Difficulties of expressing membrane proteins have been reported in number of other protein expression systems such as Escherichia coli, yeast and mammalian cell culture.  The anaerobic photoheterotroph, Rhodobacter sphaeroides, has the desirable characteristic of large quantities of intracellular membranes (typically absent in prokaryotes) as a result of the need to assemble the photosynthetic apparatus which facilitates ATP formation in light, but without oxygen formation. This growth mode can be implemented in anaerobic photobioreactors where biomass yields of more than 9.2 grams per liter dry weight (gDW/L) have been achieved.

The production of the homologous water-transport membrane protein Aquaporin Z (rAqpZ) from R. sphaeroides was utilized as a model system to develop the screening approach.  Expression levels were quantified by monitoring sample fluorescence and Western Blot densitometry (WBD).  Initially, monomeric Banana YFP (mBanana) fusion was chosen as a fluorescent tag because antibodies are commercially available and there is no overlap between the fluorescence spectrum of the protein and that of R. sphaeroides.  To demonstrate the utility of such correlation for optimizing protein expression, temperature effects were studied as a simple optimization parameter.  Ambient and optimal growth temperatures (25°C and 32°C, respectively) for Rhodobacter sphaeroides were studied under anaerobic photoheterotrophic conditions, required to induce protein expression from the puc-promoter used to drive MP expression.  Correlations between off-line sampled culture fluorescence and WBD were developed through the use of pure protein standards to demonstrate the feasibility of using fluorescence measurements as the screening method for quantifying membrane protein levels inside growing cells.

A substantially improved signal-to-noise ratio could be achieved by diluting cell suspension in bovine serum albumin (BSA) to provide for refractive index matching and adjust the relative magnitude of light scattering to absorption.  Similar trends were observed in MP production per cell at both temperatures, although the productivity was much higher at 32°C due to faster growth. This work identified several opportunities for improving this screening method towards the development of an online monitoring method to track the expression of MPs in vivo.

Although the mBanana fluorescent fusion served as an effective method for correlating membrane protein concentration, these measurements could only be taken offline.  The requirement for offline measurement results because variants of GFP (green fluorescent protein) such as mBanana are obligate aerobic fluorescent proteins and require oxygen exposure to fluoresce properly.  This presents a significant experimental constraint due to the required anaerobic growth conditions. Maturation involves folding in the presence of oxygen, during which time the fluorescence level per protein increases. Measurements of mBanana-MP fusion maturation within Rhodobacter showed that mBanana required more than 40 hours to mature (20 times its literature value). This long maturation time further precludes the use of this protein for rapid and online monitoring. Ongoing research seeks to overcome this limitation by using a new generation of facultative anaerobic fluorescent proteins based on riboflavin-binding activation.  We will present the effectiveness of using pGlow-Pp1 and pGlow-Bs1 derivatives commercially available from EvoCatal as fusion tags that will hopefully allow correlations with in vivo measurements as well as online productivity monitoring.