Date of Award

1-1-2012

Language

English

Document Type

Master's Thesis

Degree Name

Master of Science (MS)

College/School/Department

Department of Biomedical Sciences

Content Description

1 online resource (v, 49 pages) : PDF file, illustrations (some color)

Dissertation/Thesis Chair

Hongmin Li

Committee Members

Haijun Chen, Mike Fasullo, Joachim Jaeger

Keywords

Ion Channel, K2P, Kcnk, Pichia Pastoris, Protein, Potassium channels, Pichia pastoris

Subject Categories

Biology

Abstract

The KCNK (two-pore-domain K+ channel) family of potassium channels are the most widely expressed potassium channels in mammals. They are expressed in numerous organs including the brain, heart, adrenal glands, and kidneys. There are fifteen members of this family which can be further divided into six sub groups. These proteins can act as leak channels, setting the rest membrane potential of electrically excitable cells. Due to their function as modulators of excitability these proteins are under control from numerous different chemical and physical signals which include oxygen tension, pH, lipids, mechanical stretch, and neurotransmitters. These channels are not as well characterized as other potassium channels. High resolution, three dimensional structures would greatly enhance the study of the K2P proteins. Currently there are only two published crystal structures; Kcnk1 and Kcnk4. It was the goal of this project to generate soluble protein that could be used for crystallization. Initially eight channels were cloned into Escherichia coli using the Gateway® Cloning system developed by Invitrogen. However, E.coli was unable to express the protein. Pichia Pastoris was then used for expression. Five channels, Kcnk1, Kcnk 3, Kcnk5, Kcnk13 and Kcnk18. Kcnk 3 was expressed and successfully purified from P.pastoris.

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