Abstract
In a previous study, we developed an E1 monobody specific for the tumor biomarker hEphA2 [PLoS ONE (2015) 10(7): e0132976]. E1 showed potential as a molecular probe for in vitro and in vivo targeting of cancers overexpressing hEphA2. In the present study, we constructed expression vectors for E1 conjugated to optical reporters such as Renilla luciferase variant 8 (Rluc8) or enhanced green fluorescent protein (EGFP) and purified such recombinant proteins by affinity chromatography in E. coli. E1-Rluc8 and E1-EGFP specifically bound to hEphA2 in human prostate cancer PC3 cells but not in human cervical cancer HeLa cells, which express hEphA2 at high and low levels, respectively. These recombinant proteins maintained >40% activity in mouse serum at 24 h. In vivo optical imaging for 24 h did not detect E1-EGFP signals, whereas E1-Rluc8 showed tumor-specific luminescence signals in PC3 but not in HeLa xenograft mice. E1-Rluc8 signals were detected at 4 h, peaked at 12 h, and were undetectable at 24 h. These results suggest the potential of E1-Rluc8 as an EphA2-specific optical imaging agent.
Highlights
Various scaffold proteins have been developed and used for diagnostic and therapeutic purposes in many human diseases [1]
To develop the hEphA2-binding monobody conjugated to a light-emitting reporter protein, expression vectors including N-terminal E1 and C-terminal reporter genes were constructed with the pETh plasmid, yielding final recombinant proteins with a C-terminal 6×His tag
These proteins were transformed into E. coli BL21Star(DE3) and induced by isopropyl β-D-1-thiogalactopyranoside (IPTG), and the recombinant His-tagged E1-Renilla luciferase variant 8 (Rluc8) (48.3 kD) and E1-enhanced green fluorescent protein (EGFP) (38.9 kD) proteins were purified using affinity chromatography (Fig 1A)
Summary
Various scaffold proteins have been developed and used for diagnostic and therapeutic purposes in many human diseases [1]. Monobodies are small scaffold proteins (molecular weight, ~10 kDa) constructed using the human fibronectin type III (Fn3) domain [2, 3] with specificities against a wide range of target molecules conferred by variations in the amino acid sequences of their three loop regions [4, 5]. Eph receptors belong to the membrane-bound tyrosine kinase family of receptors and bind to ephrin ligands on counterpart cells [6]. Their structures comprise an N-terminal extracellular region including an ephrin binding motif, a transmembrane region, and a C-terminal cytoplasmic region including a tyrosine kinase domain [7].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
