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Expression and recovery of recombinant proteins in a novel high cell density prokaryotic system based on Ralstonia eutropha.

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Call No.	Ph.D. 2004
Author	Barnard, Gavin C.
Title	Expression and recovery of recombinant proteins in a novel high cell density prokaryotic system based on Ralstonia eutropha.
Date	2004
Abstract	Escherichia coli is the standard host for recombinant protein expression in bacteria, but suffers from some limitations (e.g. poor fermentation performance, inclusion body formation and proteolysis). A novel recombinant protein expression system, based on the bacterium Ralstonia eutropha, has previously been developed in our laboratory to overcome these limitations. In this study, soluble, active, organophosphohydrolase (OPH), a protein that is prone to inclusion body formation in E. coli, was expressed at titers greater than 10 g/L, in high cell density fermentation, using T7 RNA polymerase based amplification (a very strong viral polymerase). This represents a titer that is at least 100 fold greater than titers previously reported in E. coli for this enzyme. In this study, we developed a simple recombinant protein purification scheme, where a recombinant protein is bound in vivo to an intracellular polymer natively synthesized by R. eutropha. The cell therefore produces its own biological "affinity matrix", eliminating the need for external chromatographic protein purification. We demonstrated in vivo binding and subsequent release of essentially pure GFP (Green Fluorescent Protein) and beta-galactosidase (a multimeric, catalytic enzyme) in a single separation step. Another inexpensive protein recovery scheme is to use cellulose as an inexpensive, inert chromatography matrix. In this study, a genetic library of cellulose binding domain fusions to GFP was created and engineered into R. eutropha. Two CBD-GFP fusions were identified that both express well and bind to cellulose. Secretion of a recombinant protein into the periplasmic space greatly reduces the cost and effort of recovery and purification. Gram negative bacteria secrete very few proteins into the periplasm and even fewer into the extracellular space. Many recombinant proteins, including GFP, cannot be secreted into the bacterial periplasm via the General Secretory Pathway. In this study, GFP was successfully exported into the R. eutropha periplasm via an alternative pathway known as the Twin-Arginine Translocation (TAT) pathway.
Description	xi, 191 leaves : ill. (some col.) 28 cm.
Format	application/pdf
Advisor	Gerngross, Tillman U.
Subject	Ralstonia -- Biotechnology. Microbial biotechnology. Industrial microbiology. Recombinant proteins -- Synthesis. Recombinant Proteins -- Biosynthesis.
College	Thayer School of Engineering.
Issuing Body	Dartmouth Dissertations are made available through the Dartmouth Digital Library.
Degree	Thesis (Ph.D.)--Dartmouth College.
Copyright	All rights reserved. No part of this book may be reproduced in any form by any electronic or mechanical means without permission in writing from the author.
DOI	http://dx.doi.org/10.1349/ddlp.45

Title	Page 1
Format	application/pdf
Issuing Body	Dartmouth Dissertations are made available through the Dartmouth Digital Library.
Degree	Thesis (Ph.D.)--Dartmouth College.

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Expression and recovery of recombinant proteins in a novel high cell density prokaryotic system based on Ralstonia eutropha.

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