Expression, Purification, and Crystallization of a Cryptochrome 2 Homolog from Isodiametra pulchra
Session Title
Other Abstracts
Faculty Mentor
Jason C. Hurlbert, Ph.D.
College
College of Arts and Sciences
Department
Chemistry, Physics, Geology, & the Environment
Abstract
Cryptochrome 2 and Period 2 homologs were discovered in the transcription of Isodiametra pulchra by our collaborators Danny Stanton, and Dr. Julian Smith. That appearance could be the earliest evolutionary appearance of clock proteins. Cryptochrome (Cry) and period (Per) isoforms have been heavily researched to attain a better understanding of Cry and Per proteins interactions with each other and with other clock proteins to regulate gene expression of the clock protein. Cry2 and Per2 prevent CLOCK: BMAL1 from transcribing target genes during nighttime and show peaks during the early evening. Our goal is to determine the structure of the Cry2 homolog to determine the function. In a pET28 plasmid, we acquired expression using Escherichia coli codon optimized gene encoding Isodiametra pulchra Cry2. For purification, we ran metal chelating affinity chromatography, using the his-trap nickel column. We also performed a gel filtration to determine which fractions to concentrate, and with the concentrated protein we started to use the method hanging drop vapor diffusion to crystalize Cry2. For future work, we will continue to produce Cry2, express and purify the Per2 homologue, and perform crystallization trials on Cry2 and Per2 including the Cry2/Per2 complex.
Grant Support?
Supported by an SC-INBRE grant from the National Institute for General Medical Sciences (P20GM103499).
Start Date
15-4-2023 12:00 PM
Expression, Purification, and Crystallization of a Cryptochrome 2 Homolog from Isodiametra pulchra
Cryptochrome 2 and Period 2 homologs were discovered in the transcription of Isodiametra pulchra by our collaborators Danny Stanton, and Dr. Julian Smith. That appearance could be the earliest evolutionary appearance of clock proteins. Cryptochrome (Cry) and period (Per) isoforms have been heavily researched to attain a better understanding of Cry and Per proteins interactions with each other and with other clock proteins to regulate gene expression of the clock protein. Cry2 and Per2 prevent CLOCK: BMAL1 from transcribing target genes during nighttime and show peaks during the early evening. Our goal is to determine the structure of the Cry2 homolog to determine the function. In a pET28 plasmid, we acquired expression using Escherichia coli codon optimized gene encoding Isodiametra pulchra Cry2. For purification, we ran metal chelating affinity chromatography, using the his-trap nickel column. We also performed a gel filtration to determine which fractions to concentrate, and with the concentrated protein we started to use the method hanging drop vapor diffusion to crystalize Cry2. For future work, we will continue to produce Cry2, express and purify the Per2 homologue, and perform crystallization trials on Cry2 and Per2 including the Cry2/Per2 complex.
