Date of Graduation

5-2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Engineering (PhD)

Degree Level

Graduate

Department

Chemical Engineering

Advisor/Mentor

Robert R. Beitle, Jr.

Committee Member

Christa Hestekin

Second Committee Member

David McNabb

Third Committee Member

Edgar C. Clausen

Fourth Committee Member

Suresh K. Thallapuranam

Keywords

Pure sciences; Applied sciences; Antifungal peptide; Bio processing; Chromatography; Fed batch; Fermentation; Purification

Abstract

This work illustrates that the intelligent design of a bioprocess for the production of peptides of various length is possible by having a prior knowledge of desired product, cell line, fermentation conditions, and choices for downstream recovery. Cloning, expression, and recovery of a novel 12-mer anti-Candida peptide served as an illustrative case for a proposed Escherichia coli platform. The antifungal peptide, expressed as a fusion to GFPUV during high call density fed-batch, was recovered from cell lysate, concentrated using ion exchange chromatography, and finally cleaved with cyanogen bromide. The platform was capable of producing active peptide capable of arresting the growth of yeast, used as an indicator strain. During processing, a new E. coli cell line (Lotus®) was used for expression to investigate the effect of certain mutations that reduced the downstream burden of host cell proteins (HCPs). A 37% improvement in initial capture efficiency of diethylaminoethyl (DEAE) resin was observed and attributed to less HCPs. To further streamline downstream purification, a mutant of GFPUV was designed that was resistant to cleavage using cyanogen bromide. Finally, an economic analysis demonstrated the positive economic implications of the suggested improvements.

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