Bartlow, Patrick
(2012)
DESIGN OF ESCHERICHIA COLI HOST STRAINS FOR IMPROVED
RECOMBINANT PROTEIN PURIFICATION: AN APPROACH THAT BRIDGES THE
UPSTREAM AND DOWNSTREAM REALMS OF BIOPROCESSING.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Escherichia coli is a favored host for rapid, scalable expression of recombinant proteins for academic, commercial or therapeutic use. To maximize its economic advantages, however, it must be coupled with robust downstream processes. These are typically composed of three or fewer chromatography steps that remove the majority of the host proteins to achieve a reasonable degree of purification. Removal of remaining impurities is often very difficult and costly due similarity among physicochemical properties with the target. This work implements a novel approach that overcomes such limitations by bridging upstream and downstream realms, that is, by subjecting lysed cell material to a variety of purification procedures, identifying individual impurities and mitigating their removal by genetic modification.
Successful knockout of three prominent contaminants of immobilized metal affinity chromatography (IMAC) without detriment to cell growth or recombinant protein expression was demonstrated. Elution of the recombinant target was strategically manipulated with peptide tags that allowed purification to virtual homogeneity. Additional IMAC studies focused on the most problematic host proteins, those that retained binding affinity under stringent conditions. Two-dimensional difference gel electrophoresis discerned variation in the soluble extract pools loaded in IMAC and the subsequent impurities, with respect to varied levels of recombinant protein expression. Peptidyl-prolyl isomerase SlyD and catabolite activator protein were shown to be the most persistent contaminants and had greater prevalence at low target protein expression. Since genetic removal of the transcription activator would negatively impact cellular function, I substituted specific residues to eliminate its IMAC affinity with minimal impact on its activity.
I applied this integrative strategy seeking to improve performance of cheaper, non-affinity based processes. Phosphoenolpyruvate carboxykinase and peptidase D were significant contaminants during serial purification of a target by hydrophobic interaction and anion exchange chromatography. Ribosomal protein L25 dominated non-target binding of a polyarginine tagged recombinant on cation exchange resin.
With the development of comprehensive genomic manipulation in higher order species, such integrative approaches will be conventional in the development of coupled expression systems for the production of complex biologics.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
2 February 2012 |
| Date Type: |
Publication |
| Defense Date: |
26 September 2011 |
| Approval Date: |
2 February 2012 |
| Submission Date: |
1 December 2011 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
146 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Swanson School of Engineering > Bioengineering |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
Escherichia coli
Strain optimization
Protein purification
Immobilized metal affinity
Chromatography |
| Date Deposited: |
02 Feb 2012 14:37 |
| Last Modified: |
19 Dec 2016 14:38 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/10617 |
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