Abstract
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.
Highlights
The centrosome is the main microtubule organizing center (MTOC) in animal cells
In non-mitotic interphase cells, the centrosome is located near the nucleus to produce an assembly of microtubules that radiates towards the cell periphery, serving as tracks for motor protein-mediated transport of cellular compartments
The centrosome consists of a pair of orthogonally arranged centrioles surrounded by pericentriolar material (PCM)
Summary
In non-mitotic interphase cells, the centrosome is located near the nucleus to produce an assembly of microtubules that radiates towards the cell periphery, serving as tracks for motor protein-mediated transport of cellular compartments. Pericentrin (PCNT) is a large ∼360 kDa protein that exists within. PCNT contains a series of predicted coiled-coil regions over most of their length [5], but a highly conserved PCM targeting motif called the PACT domain is found near the C-terminus [7] (Figure 1). Three regions in the human PCNT which are predicted as coiled-coils were successfully over-produced in Escherichia coli using plasmids containing E. coli codon-optimized PCNT gene. 1. Regions of human pericentrin (PCNT) showing predicted coiled-coil (CC) and the PACT
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