Molecular and Genetic Strategies to Enhance Functional Expression of Recombinant Protein in Escherichia coli
The versatile Escherichia coli facilitates protein expression with relative simplicity, high cell density on inexpensive substrates, well known genetics, variety of expression vectors, mutant strains, co-overexpression technology, extracytoplasmic secretion systems, and recombinant protein fusion pa...
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2009
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ftunivwaterloo:oai:uwspace.uwaterloo.ca:10012/4721 2023-05-15T13:43:38+02:00 Molecular and Genetic Strategies to Enhance Functional Expression of Recombinant Protein in Escherichia coli Narayanan, Niju 2009 http://hdl.handle.net/10012/4721 en eng University of Waterloo http://hdl.handle.net/10012/4721 Escherichia coli recombinant protein expression cell-surface display protein secretion inclusion bodies proteolysis mutagenesis chaperone fusion tags cell physiology stress pathways Chemical Engineering Doctoral Thesis 2009 ftunivwaterloo 2022-06-18T22:58:36Z The versatile Escherichia coli facilitates protein expression with relative simplicity, high cell density on inexpensive substrates, well known genetics, variety of expression vectors, mutant strains, co-overexpression technology, extracytoplasmic secretion systems, and recombinant protein fusion partners. Although, the protocol is rather simple for soluble proteins, heterologous protein expression is frequently encountered by major technical limitations including inefficient translation, formation of insoluble inclusion bodies, lack of posttranslational modification mechanisms, degradation by host proteases, and impaired cell physiology due to host/protein toxicity, in achieving functional expression of stable, soluble, and bioactive protein. In this thesis, model protein expression systems are used to address the technical issues for enhancing recombinant protein expression in E. coli. When yellow fluorescence protein (YFP) was displayed on E. coli cell surface, the integrity of the cell envelope was compromised and cell physiology was severely impaired, resulting in poor display performance, which was restored by the coexpression of Skp, a periplasmic chaperone. On the basis of monitoring the promoter activities of degP, rpoH, and cpxP under various culture conditions, it was demonstrated that the cell-surface display induced the σE extracytoplasmic stress response, and PdegP::lacZ was proposed to be a suitable “sensor” for monitoring extracytoplasmic stress. Intracellular proteolysis has been recognized as one of the key factors limiting recombinant protein production, particularly for eukaryotic proteins heterologously expressed in the prokaryotic expression systems of E. coli. Two amino acids, Leu149 and Val223, were identified as proteolytically sensitive when Pseudozyma antarctica lipase (PalB) was heterologously expressed in Escherichia coli. The functional expression was enhanced using the double mutant for cultivation. However, the recombinant protein production was still limited by PalB misfolding, ... Doctoral or Postdoctoral Thesis Antarc* Antarctica University of Waterloo, Canada: Institutional Repository |
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Open Polar |
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University of Waterloo, Canada: Institutional Repository |
op_collection_id |
ftunivwaterloo |
language |
English |
topic |
Escherichia coli recombinant protein expression cell-surface display protein secretion inclusion bodies proteolysis mutagenesis chaperone fusion tags cell physiology stress pathways Chemical Engineering |
spellingShingle |
Escherichia coli recombinant protein expression cell-surface display protein secretion inclusion bodies proteolysis mutagenesis chaperone fusion tags cell physiology stress pathways Chemical Engineering Narayanan, Niju Molecular and Genetic Strategies to Enhance Functional Expression of Recombinant Protein in Escherichia coli |
topic_facet |
Escherichia coli recombinant protein expression cell-surface display protein secretion inclusion bodies proteolysis mutagenesis chaperone fusion tags cell physiology stress pathways Chemical Engineering |
description |
The versatile Escherichia coli facilitates protein expression with relative simplicity, high cell density on inexpensive substrates, well known genetics, variety of expression vectors, mutant strains, co-overexpression technology, extracytoplasmic secretion systems, and recombinant protein fusion partners. Although, the protocol is rather simple for soluble proteins, heterologous protein expression is frequently encountered by major technical limitations including inefficient translation, formation of insoluble inclusion bodies, lack of posttranslational modification mechanisms, degradation by host proteases, and impaired cell physiology due to host/protein toxicity, in achieving functional expression of stable, soluble, and bioactive protein. In this thesis, model protein expression systems are used to address the technical issues for enhancing recombinant protein expression in E. coli. When yellow fluorescence protein (YFP) was displayed on E. coli cell surface, the integrity of the cell envelope was compromised and cell physiology was severely impaired, resulting in poor display performance, which was restored by the coexpression of Skp, a periplasmic chaperone. On the basis of monitoring the promoter activities of degP, rpoH, and cpxP under various culture conditions, it was demonstrated that the cell-surface display induced the σE extracytoplasmic stress response, and PdegP::lacZ was proposed to be a suitable “sensor” for monitoring extracytoplasmic stress. Intracellular proteolysis has been recognized as one of the key factors limiting recombinant protein production, particularly for eukaryotic proteins heterologously expressed in the prokaryotic expression systems of E. coli. Two amino acids, Leu149 and Val223, were identified as proteolytically sensitive when Pseudozyma antarctica lipase (PalB) was heterologously expressed in Escherichia coli. The functional expression was enhanced using the double mutant for cultivation. However, the recombinant protein production was still limited by PalB misfolding, ... |
format |
Doctoral or Postdoctoral Thesis |
author |
Narayanan, Niju |
author_facet |
Narayanan, Niju |
author_sort |
Narayanan, Niju |
title |
Molecular and Genetic Strategies to Enhance Functional Expression of Recombinant Protein in Escherichia coli |
title_short |
Molecular and Genetic Strategies to Enhance Functional Expression of Recombinant Protein in Escherichia coli |
title_full |
Molecular and Genetic Strategies to Enhance Functional Expression of Recombinant Protein in Escherichia coli |
title_fullStr |
Molecular and Genetic Strategies to Enhance Functional Expression of Recombinant Protein in Escherichia coli |
title_full_unstemmed |
Molecular and Genetic Strategies to Enhance Functional Expression of Recombinant Protein in Escherichia coli |
title_sort |
molecular and genetic strategies to enhance functional expression of recombinant protein in escherichia coli |
publisher |
University of Waterloo |
publishDate |
2009 |
url |
http://hdl.handle.net/10012/4721 |
genre |
Antarc* Antarctica |
genre_facet |
Antarc* Antarctica |
op_relation |
http://hdl.handle.net/10012/4721 |
_version_ |
1766191345956814848 |