Cloning, Fed-Batch Expression And Purification Of A Novel Anti-Candida Peptide And Development Of A Cleavage Resistant Variant Of Green Fluorescence Protein
Date of Graduation
Doctor of Philosophy in Engineering (PhD)
Robert R. Beitle, Jr.
Second Committee Member
Third Committee Member
Edgar C. Clausen
Fourth Committee Member
Suresh K. Thallapuranam
Pure sciences; Applied sciences; Antifungal peptide; Bio processing; Chromatography; Fed batch; Fermentation; Purification
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.
Mukherjee, R. (2016). Cloning, Fed-Batch Expression And Purification Of A Novel Anti-Candida Peptide And Development Of A Cleavage Resistant Variant Of Green Fluorescence Protein. Graduate Theses and Dissertations Retrieved from https://scholarworks.uark.edu/etd/1597