Molecular engineering of an EGFP/disintegrin-based integrin marker.
Disintegrins are viper venom peptides, which bind integrins with high affinity (10(-8) M) and selectivity. Among them, eristostatin (Er) selectively binds and inhibits alphaIIbbeta3 integrin function. In this work we have engineered an enhanced green fluorescence protein (EGFP)-tagged Er as an alphaIIbbeta3 biomarker to be used in bioassays involving fluorescence detectors. For this, we have first constructed an EGFP bacterial expression vector, which resulted in a 6xHis tag-coding region followed by the EGFP gene and a 3' multiple cloning site (MCS) comprising nine restriction sites. This vector, termed pAZ, was used to clone the Er gene, resulting in a 32 kDa EGFP-Er fusion protein when expressed as characterized by SDS-PAGE and Western blot. Both EGFP-Er and EGFP (expressed from the empty pAZ vector) were purified by immobilized metal affinity chromatography (IMAC) and their fluorescence was measured showing similar values, thus suggesting that the Er portion is not affecting the EGFP activity. EGFP-Er, but not EGFP selectively bound to immobilized platelets as detected by confocal microscopy indicating the preservation of Er disintegrin activity and its potential use as a marker for alphaIIbbeta3 integrin. Our data suggest the use of the pAZ vector for expressing soluble EGFP-labeled proteins and the use of EGFP-fused disintegrins as markers for integrins.
Butera
Diego
D
Laboratório de Imunopatologia, Instituto Butantan, Av. Vital Brasil, 1500, 05503-900 São Paulo, Brazil. dbutera@butantan.gov.br
Piazza
Roxane Maria Fontes
RM
McLane
Mary Ann
MA
Chammas
Roger
R
da Silva
Ana Maria Moura
AM
D000818:Animals; D015415:Biomarkers; D001792:Blood Platelets / Q000378:metabolism; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D019483:Disintegrins / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D005818:Genetic Engineering; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D018613:Microscopy, Confocal; D010455:Peptides / Q000378:metabolism; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism; D014757:Viper Venoms / Q000737:chemistry* / Q000378:metabolism
D015415:Biomarkers; D019483:Disintegrins; D010455:Peptides; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D014757:Viper Venoms; C485184:enhanced green fluorescent protein; C084060:eristostatin; D049452:Green Fluorescent Proteins