Heterologous protein expression in photosynthetic bacteria and its application in expressing cytochrome component from an electron transfer pathway

Abstract

The photosynthetic microorganisms, Rhodobacter species, have attracted increasing interests as expression host in bioproduction. Their unique metabolism has made these bacteria suitable for producing membrane proteins and proteins modified with metal cofactors. Meanwhile, their ability to utilize solar energy offers attractive benefits which cannot be enjoyed by traditional hosts, e.g. E. coli. In recent years, structural biologists have been using Rhodobacter sphaeroides for the production of membrane proteins. In this thesis research, we aim to provide a detailed characterization study on Rhodobacter sphaeroides 2.4.1 as an expression host for heterologous proteins and to explore its usage in the expression of a cytochrome component protein (i.e., MtrA). The thesis work started with the expression of MtrA protein, which involves in an extracellular electron transfer pathway in Shewanella oneidensis, in Rhodobacter sphaeroides 2.4.1. Using the expression vector, pIND4, MtrA protein was successfully expressed in Rhodobacter sphaeroides 2.4.1 and confirmed by Western blotting. The intracellular location of MtrA was verified by periplasmic extraction in the preparation of Western blotting samples. The function of MtrA was also verified through ferrozine assay by quantifying the ability to reduce ferric ions in Rhodobacter sphaeroides 2.4.1 expressing MtrA. To facilitate the function of MtrA in a complete electron transfer pathway, other components in an MtrCAB pathway need to be expressed. Thus a multi-protein expression vector is needed. In order to construct a bicistronic expression vector for the Rhodobacter system, the vector pRK442(H) was chosen as a vector backbone. The expression system constructed on the basis of this backbone suffers from the phototoxicity of tetracycline due to its usage for maintaining the plasmid under phototrophic condition. To overcome this difficulty, pHW1 was constructed using the protein expression cassette on an inducible plasmid, pIND4, and ampicillin resistant gene was used to replace the native tetracycline resistant gene carried by pRK442(H). After the construction of pHW1, features about this plasmid were characterized including the segregational plasmid stability, protein expression level and so on. The data showed that anaerobic culture, dark condition and relatively high inducer concentration enhanced the plasmid stability. The effect of different stages at which IPTG induction was performed on protein expression level was also explored. This part is a component of a project to construct a novel photosynthetic micro fuel cell. Now the focus is mainly on construct electron pathway. The first component has been expressed and new plasmid pRKBic without phototoxicity problem will be used in the expressing of multi-cytochromes from Shewanella oneidensis in Rhodobacter sphaeroides 2.4.1.

Date of Award	2013
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology