Data for the inhibition effects of recombinant lamprey CRBGP on the tube formation of HUVECs and new blood vessel generation in CAM models

In the present data article, lamprey cysteine-rich buccal gland protein (CRBGP) which belongs to cysteine-rich secretory proteins (CRISPs) family was recombinant and expressed in Rosetta blue cells. After identification, the recombinant protein was purified through affinity chromatograph. The inhibition effects of recombinant lamprey CRBGP (rL-CRBGP) on tube formation of human umbilical vein endothelial cells (HUVECs) and new blood vessel generation in chick chorioallantoic membrane (CAM) models were analyzed. This paper contains data related to research concurrently published in “Anti-angiogenic activities of CRBGP from buccal glands of lampreys (Lampetra japonica)” [1].


a b s t r a c t
In the present data article, lamprey cysteine-rich buccal gland protein (CRBGP) which belongs to cysteine-rich secretory proteins (CRISPs) family was recombinant and expressed in Rosetta blue cells. After identification, the recombinant protein was purified through affinity chromatograph. The inhibition effects of recombinant lamprey CRBGP (rL-CRBGP) on tube formation of human umbilical vein endothelial cells (HUVECs) and new blood vessel generation in chick chorioallantoic membrane (CAM) models were analyzed. This paper contains data related to research concurrently published in "Anti-angiogenic activities of CRBGP from buccal glands of lampreys (Lampetra japonica)" [1]. &

Data
As shown in Fig. 1, lamprey CRBGP is a very conservative gene and has 45% sequence identity with the ES-CRISP from the snake venom of Echis carinatus sochureki. Subsequently, lamprey CRBGP was subcloned into a pEGX-4T-1 vector and expressed as a Glutathione S-transferase (GST)-tagged fusion protein in Rosetta blue cells with the molecular weight of 51.6 kDa (Fig. 2). After identification by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF/TOF) analysis, rL-CRBGP was found to exhibit the anti-angiogenic activities in both tube formation and CAM assays (Figs. 3-5).

Sequence alignment
Additional 10 CRISP sequences from the other species were obtained from ExPASy (http://www. expasy.ch/tools/blast). The multiple sequence alignments of CRISPs were performed by ClustalX (1.81) software using default settings [2].

Expression, purification, and identification of rL-CRBGP
A pair of PCR primers (CRBGP-F: 5 0 -CCGGAATTCGCGAGCGTCGTGGCGGCGACA-3 0 ; CRBGP-R: 5 0 -AGAAGAATGCGGCCGCCTGCACATCCGTCG-3 0 ) was designed based on the sequence of lamprey CRBGP [3], flanked by an EcoR I and a Not I restriction site. Lamprey CRBGP was amplified and subcloned into a pEGX-4T-1 vector with a GST-tag. rL-CRBGP was expressed in Rosetta blue cells induced with 1 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) for 36 h. The cells were collected by centrifugation, and washed in PBS for twice (pH 7.4). Subsequently, the cells were resuspended in the PBS (pH 7.4) and sonicated on ice for 60 min. After centrifugation, the soluble supernatant was collected and subjected to a GSTrap TM 4B column (GE, USA) equilibrated with binding buffer (PBS, pH 7.4). After washing the column with wash buffer (PBS, pH 7.4), the rL-CRBGP was collected in elution buffer containing 50 mM Tris-HCl, 20 mM glutathione (pH 8.0). The concentration of rL-CRBGP was also measured using a bicinchoninic acid (BCA) protein assay Kit (Beyotime Biotechnology, China). The purified rL-CRBGP was analyzed by 12% SDS-PAGE and stained with Coomassie brilliant blue R-250. The rL-CRBGP was digested with trypsin (25 mM, Promega) in-gel overnight and identified by MALDI-TOF/TOF mass spectrometry (Bruker, USA).

Anti-angiogenic activity assay of rL-CRBGP
Similar to that of native lamprey CRBGP, the anti-angiogenic activity of rL-CRBGP was also performed in both tube formation assay and CAM models according to the methods reported by Qi Jiang and colleagues [1].