Recombinant expression in human cells of active integrin alpha 1 beta 1-blocking RTS-disintegrin jerdostatin.
Jerdostatin, an RTS short disintegrin cloned from Protobothrops jerdonii and recombinantly produced in Escherichia coli, is a potent and specific antagonist of the alpha(1)beta(1) integrin. Jerdostatin selectively blocked the adhesion of alpha(1)beta(1)-K562 cell to collagens I and IV in vitro and angiogenesis in vivo. Here we report the recombinant production of jerdostatin in a mammalian cell system, a prerequisite for developing a conditional transgenic mouse to investigate the effect of systemic expression of jerdostatin on tumor development. For proper export of jerdostatin, a secretion leader sequence was engineered at the protein's N-terminus. A FLAG epitope was also included at the N-terminus of the mature disintegrin to facilitate its isolation and characterization of recombinant jerdostatin (rJerd). This pRc-CMV/FLAG-rJerd construct was transiently expressed in HEK-293 cells and was efficiently secreted into the culture medium. rJerd bound to recombinant soluble alpha(1)beta(1) integrin in a saturable and cation-independent manner. Soluble rJerd also inhibited the binding of alpha(1)beta(1) integrin to the CB3 fragment of collagen IV in a dose-dependent manner (IC(50) 570 nM). Mammalian cell-expressed jerdostatin disrupted the adhesion of RuGli cells to collagen IV. Our results highlight pRc-CMV/FLAG-rJerd as a suitable construct for expressing soluble active alpha(1)beta(1)-blocking jerdostatin in a mammalian cell system.
Juárez
Paula
P
Instituto de Biomedicina de Valencia, CSIC, Jaime Roig 11, 46010 Valencia, Spain.
Bolás
Gema
G
de Rezende
Flavia Figueiredo
FF
Calvete
Juan J
JJ
Eble
Johannes A
JA
D000595:Amino Acid Sequence; D001483:Base Sequence; D024141:Collagen Type IV / Q000378:metabolism; D019483:Disintegrins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D000939:Epitopes / Q000378:metabolism; D005818:Genetic Engineering; D057809:HEK293 Cells; D006801:Humans; D039222:Integrin alpha1beta1 / Q000037:antagonists & inhibitors*; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics; D010975:Platelet Aggregation Inhibitors / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D014162:Transfection
D024141:Collagen Type IV; D019483:Disintegrins; D000939:Epitopes; D039222:Integrin alpha1beta1; D009842:Oligopeptides; D010455:Peptides; D010975:Platelet Aggregation Inhibitors; D011994:Recombinant Proteins; C554060:jerdostatin, Protobothrops jerdonii; C085706:FLAG peptide