Expression, crystallization and preliminary crystallographic analysis of C-reactive protein from zebrafish

Abstract

C-reactive protein (CRP) is an acute phase protein that is found in blood, the concentration of which in plasma rises rapidly in response to inflammation. It functions as a pattern-recognition molecule, recognizing dead cells and various pathogenic agents and eliminating them by utilizing the classical complement pathway and activating macrophages. CRP is phylogenetically highly conserved in invertebrates and mammals. To date, information on the CRP gene has been reported from numerous species of animals, but little is known about the structure of CRP from species other than humans. In order to solve the structure of CRP from bony fish, the CRP gene from zebrafiah (Danio rerio) was cloned and expressed in Escherichia coli. The zebrafish CRP (Dare-CRP) was then purified and crystallized. The crystal diffracted to 2.3 Å resolution and belonged to space group R3, with unit-cell parameters a = b = 114.7, c = 61.0 Å. The Matthews coefficient and solvent content were calculated to be 3.28 Å(3) Da(-1) and 62.55%, respectively. Determination of the zebrafish CRP structure should be helpful in investigating the evolution of CRPs in the innate immune system.

Evolution of C-Reactive Protein

“…Dogfish CRP exhibits immunological cross-reactivity with rabbit CRP. The CRP gene from zebrafish, Danio rerio , a bony fish, has been cloned, expressed, protein purified, and crystallized (37, 38, 74, 75). There are seven CRP-like genes in zebrafish which are differentially expressed both normally and in acute phase and have anti-viral activities.…”

Crystal structures for short-chain pentraxin from zebrafish demonstrate a cyclic trimer with new recognition and effector faces

“…The Dare-PTX gene was amplified by PCR as previously described (Chen et al, 2011). The gene encoding Dare-PTX was further subcloned into the expression vector pET-21a using the restriction enzymes EcoR I and Xho I.…”

Characterization of the innate immune response to Streptococcus pneumoniae infection in zebrafish

“…The analysis at the amino acid level shows that all the zebrafish Crps show equal similarity (31-34%) to the human CRP (Comparisons done by Clustal Omega [149,150]). Despite this relatively low-level similarity, the overall structure, including the ligand binding pocket as well as binding sites for C1q and Fc receptor, is relatively well conserved in zebrafish [151][152][153]. As the functionality of the zebrafish Crps has not been analyzed in detail, it is not possible to assign functional homology, although the existence of this protein family across species and its retained ability to activate complement in fish suggests that it serves a similar function in zebrafish [154,155].…”