Characterization and application of a periplasmic protein releasing system for extracellular recombinant protein production in Escherichia coli

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dc.contributor.advisorSan, Ka-Yiuen_US
dc.creatorYu, Pengen_US
dc.date.accessioned2009-06-04T00:03:08Zen_US
dc.date.available2009-06-04T00:03:08Zen_US
dc.date.issued1993en_US
dc.description.abstractEscherichia coli is commonly used as a host in the production of high value therapeutic proteins. However, one major drawback associated with E. coli being a host is the intracellular location of product proteins. As a result, mechanical cell disruption is commonly employed in biotechnology industry for protein release. Unfortunately, mechanical disintegration always contaminates the target protein with other cellular substances. On the other hand, progress in the study of protein translocation across membrane often allows the transport of proteins from the cytoplasm to the periplasm. However, the outer membrane still presents a barrier for their extracellular release. The objective of this work is to bridge the gap between protein translocation and extracellular protein release. The main focus is to characterize a periplasmic protein release system and to apply the system to different processes for extracellular protein production. Bacteriocin Release Protein (BRP) and glycine were the two systems studied using a-amylase as the model protein. Both agents were shown to be effective in periplasmic protein release. When used alone in a batch fermentor, BRP released 41% of the periplasmic a-amylase extracellularly without causing substantial cell lysis. Similarly, glycine achieved a 45% protein release. Synergistic combination of BRP and glycine resulted in further increase in a-amylase release. At an optimal combination, 78% of a-amylase was released. Application of the release system for continuous extracellular protein production was successful. A maximum productivity of 8.3 units/ml/hr was obtained, of which one third was in the extracellular broth. Mathematical simulation suggested that higher expression levels of a-amylase at slower growth rates is mainly due to a higher plasmid copy number. Incorporation of hollow fiber cartridges into a chemostat process allowed continuous production of cell-free proteins as well as retention of recombinant cells. At an optimal dilution rate, 42% of a-amylase was produced in a cell-free form. Substitution of the Amp$\sp{\rm r}$ gene in pBR322-amy with a Km$\sp{\rm r}$ gene in an attempt to improve the performance of the release system resulted in a higher accumulation of a-amylase in the cytoplasm. The observation demonstrated that antibiotic markers are important parameters in a-amylase translocation and distribution.en_US
dc.format.extent169 p.en_US
dc.format.mimetypeapplication/pdfen_US
dc.identifier.callnoThesis Ch.E. 1993 Yuen_US
dc.identifier.citationYu, Peng. "Characterization and application of a periplasmic protein releasing system for extracellular recombinant protein production in Escherichia coli." (1993) Diss., Rice University. <a href="https://hdl.handle.net/1911/16694">https://hdl.handle.net/1911/16694</a>.en_US
dc.identifier.urihttps://hdl.handle.net/1911/16694en_US
dc.language.isoengen_US
dc.rightsCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.en_US
dc.subjectChemical engineeringen_US
dc.subjectMolecular biologyen_US
dc.subjectMicrobiologyen_US
dc.subjectBiologyen_US
dc.subjectEngineeringen_US
dc.titleCharacterization and application of a periplasmic protein releasing system for extracellular recombinant protein production in Escherichia colien_US
dc.typeThesisen_US
dc.type.materialTexten_US
thesis.degree.departmentChemical Engineeringen_US
thesis.degree.disciplineEngineeringen_US
thesis.degree.grantorRice Universityen_US
thesis.degree.levelDoctoralen_US
thesis.degree.nameDoctor of Philosophyen_US
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