Over-Production, Crystallization, and Preliminary X-ray Crystallographic Analysis of a Coiled-Coil Region in Human Pericentrin

• Main Authors: Min Ye Kim, Jeong Kuk Park, Yeowon Sim, Doheum Kim, Jeong Yeon Sim, SangYoun Park
• Format: Article
• Language: English
• Published: MDPI AG 2017-10-01
• Series: Crystals
• Subjects: pericentrin; coiled-coil; centrosome; pericentriolar material (PCM)
• Online Access: https://www.mdpi.com/2073-4352/7/10/296

The genes encoding three coiled-coil regions in human pericentrin were gene synthesized with Escherichia coli codon-optimization, and the proteins were successfully over-produced in large quantities using E. coli expression. After verifying that the purified proteins were mostly composed of α-helices, one of the proteins was crystallized using polyethylene glycol 8000 as crystallizing agent. X-ray diffraction data were collected to 3.8 Å resolution under cryo-condition using synchrotron X-ray. The crystal belonged to space group C2 with unit cell parameters a = 324.9 Å, b = 35.7 Å, c = 79.5 Å, and β = 101.6˚. According to Matthews’ coefficient, the asymmetric unit may contain up to 12 subunits of the monomeric protein, with a crystal volume per protein mass (VM) of 1.96 Å3 Da−1 and a 37.3% solvent content.