Project Title

Experimental Protocol for the Production of Bacterial Expressed CTRP3

Authors' Affiliations

Sydney Keith and Dr. Jonathan Peterson, Department of Health Sciences, College of Public Health, East Tennessee State University, Johnson City, Tennessee

Location

Ballroom

Start Date

4-5-2018 8:00 AM

End Date

4-5-2018 12:00 PM

Poster Number

47

Name of Project's Faculty Sponsor

Jonathan Peterson

Faculty Sponsor's Department

Health Sciences

Type

Poster: Competitive

Classification of First Author

Undergraduate Student

Project's Category

Biomedical and Health Sciences

Abstract Text

INTRODUCTION: Previous studies have shown that mammalian expressed CTRP3 functions to lower blood glucose levels in transgenic mice. However, the production of mammalian expressed CTRP3 is expensive and can only yield up to 1mg/L of recombinant protein. On the contrary, bacterial expressed CTRP3 is relatively inexpensive and can theoretically produce 70mg of recombinant protein per L of bacteria. The purpose of this experiment is to see if functional CTRP3 protein can be produced effectively and economically using bacterial expression system. METHODS: The ctrp3 gene was inserted into a bacterial expression plasmid vector and transfected into Escherichia Coli (E. coli) bacterium to produce recombinant Polyhistidine-tagged-CTRP3 in the presence of L-Arabinose. Optimal expression parameters were then tested in the transfected bacteria. Optimal expression was confirmed by measurement of CTRP3 in through immunoblotting. RESULTS: Once optimal expression conditions were established, large amounts of CTRP3 were generated and purified by commercial HIS purification systems. Due to post-translation modification of CTRP3, it is unclear if the purified CTRP3 will be functional. The next step in the process is to optimize the purification protocol and test the functionality of the bacterial expressed CTRP3.

Support or Funding Information

This research was supported in part by National Institute on Alcohol Abuse and Alcoholism of the National Institutes of Health under Award Number R03AA023612 and East Tennessee State University Research Development Committee (E82262).

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Apr 5th, 8:00 AM Apr 5th, 12:00 PM

Experimental Protocol for the Production of Bacterial Expressed CTRP3

Ballroom

INTRODUCTION: Previous studies have shown that mammalian expressed CTRP3 functions to lower blood glucose levels in transgenic mice. However, the production of mammalian expressed CTRP3 is expensive and can only yield up to 1mg/L of recombinant protein. On the contrary, bacterial expressed CTRP3 is relatively inexpensive and can theoretically produce 70mg of recombinant protein per L of bacteria. The purpose of this experiment is to see if functional CTRP3 protein can be produced effectively and economically using bacterial expression system. METHODS: The ctrp3 gene was inserted into a bacterial expression plasmid vector and transfected into Escherichia Coli (E. coli) bacterium to produce recombinant Polyhistidine-tagged-CTRP3 in the presence of L-Arabinose. Optimal expression parameters were then tested in the transfected bacteria. Optimal expression was confirmed by measurement of CTRP3 in through immunoblotting. RESULTS: Once optimal expression conditions were established, large amounts of CTRP3 were generated and purified by commercial HIS purification systems. Due to post-translation modification of CTRP3, it is unclear if the purified CTRP3 will be functional. The next step in the process is to optimize the purification protocol and test the functionality of the bacterial expressed CTRP3.

Support or Funding Information

This research was supported in part by National Institute on Alcohol Abuse and Alcoholism of the National Institutes of Health under Award Number R03AA023612 and East Tennessee State University Research Development Committee (E82262).