eCommons will be completely unavailable from 8:00am April 4 until 5:00pm April 5, 2018, for software upgrades. Thank you for your patience during this planned service interruption. Please contact us at firstname.lastname@example.org if you have questions or concerns.
Enhancing Protein Secretion In Escherichia Coli By Codon Engineering Via Translation Optimization And Genome Sequence Analysis Of A Hypersecreter Mutant
Escherichia coli is a common host for recombinant protein production for biotechnology applications. Secretion of recombinant proteins to the extracellular and periplasmic space has the potential to reduce protein aggregation and to simplify downstream purification. A directed mutagenesis approach (specifically changing abundant codons to synonymous rare codons in a specific region) previously resulted in an eight-fold improvement in active hemolysin (HlyA) secretion. Also, synonymous codon substitutions have been shown to alter protein folding and function; however this mechanism is not well understood. In the first part of this study, a series of experiments have been described to study the effect of synonymous rare codon clusters on protein folding and secretion via multiple pathways in E. coli. Significant improvement in extracellular and periplasmic secretion of various recombinant proteins was observed by synonymous rare codon engineering. The analyses also revealed that synonymous rare codon cluster at specific sites of the target gene can alter polypeptide folding and activity by modulating the interactions of polypeptide with molecular chaperones. The study provides an experimental toolkit to enhance recombinant protein secretion in E. coli and offer insights into the effect of silent mutations on protein folding. The second part of the thesis includes genome sequence analysis of a derivative hypersecreter E. coli strain (B41), created previously by random mutagenesis, and the parent strain using a 'next-generation' sequencing technology. Mutational profiling revealed a single nucleotide polymorphism (G in the B41 genome, which results in premature translation termination of a transcription factor, RutR. Comparative mRNA expression analysis revealed that absence of RutR coordinates a decrease in the expression of tRNA-synthetases and some amino acid transporter genes, suggesting that the absence of RutR may result in slower translation rate. The work presents a single gene target to enhance extracellular secretion via the Type-I pathway and highlight the potential of new high-throughput massively parallel sequencing technologies to characterize selected mutants for strain improvement.
dissertation or thesis