Date of Award

2014

Degree Type

Thesis

Department

Chemical and Biomedical Engineering

First Advisor

Holland, Nolan

Subject Headings

Protein engineering, Recombinant proteins, Polypeptides, Escherichia coli, Antifreeze proteins, Biomedical engineering

Abstract

Fusion protein technologies can aid to improve solubility of recombinant protein from microorganisms and help recombinant protein purification. Elastin-like polypeptides (ELP) as a fusion tag can be utilized to facilitate the purification of recombinant proteins because ELP can provide its thermally responsive behavior to ELP tagged proteins. An ELP tag can be used as the purification carrier through inverse transition cycling (ITC), which is a simple and non chromatographic separation process. The purification through ITC can reduce cost and can be quickly performed compared to other purification methods. However, we further considered ELP tag removal because it may possibly hinder the structure and function of fusion partners (or target proteins). Tenebrio molitor antifreeze protein (TmAFP) is an insect AFP and it is classified as a hyperactive antifreeze protein because its activity is 10 to 100 times greater than AFPs from other sources. TmAFP as a target protein has been purified by cold finger purification or chromatographic separation but there are drawbacks. The overall goal of this work is to design and implement a strategy for high yield of recombinant TmAFP in E. coli through purification using ELP tags. This includes ELP tagged TmAFP with a TEV cleavage site. DNA encoding this tag was designed and successfully added to the N-terminus of the TmAFP gene. Then the ELP tags were added to the N-terminus of TmAFP and ELP tag DNA was successfully added to N-terminus of TEV protease. Of note, ELP[(GVGVP)40] tagged DNA was successfully added to the N-terminus TEV protease but ELP[(GVGVP)20] tagged DNA was not successfully added. Then the ELP tagged fusion proteins were successfully expressed. ELP tagged TmAFPs were successfully purified by using ITC while ELP tagged TEV protease was not successfully purified by ITC

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