title: Purification and characterization of a novel cell-penetrating carrier similar to cholera toxin chimeric protein.
abstract: Developing a recombinant vector for noninvasively delivering biological macromolecules into the brain is important. This study constructed and purified a protein complex based on the cholera toxin (CT) molecular structure. Enhanced green fluorescent protein (EGFP)-modified A2 subunits of CT (CTA2) were used as tracer molecules for introduction of transactivator of transcription (TAT) through the A subunit into cells. The protein complex EGFP-CTA2-TAT/(CTB)5 (CTB: B subunit of CT) was obtained using an in vitro recombination method and verified by monosialoganglioside-enzyme-linked immunosorbent assay and high performance liquid chromatography assay. The protein complexes bound more strongly to monosialoganglioside (GM1) than (CTB)5 at low concentrations (0.625-1.25 μg/mL). In vitro assays revealed that the transmembrane function of TAT was also maintained. The GM1-binding activity and cell membrane-penetrating ability suggested that a CT structure-based protein complexes could be used to design a delivery carrier for intranasal administration through GM1 binding. The expression vector introduced in this study provides a feasible expression frame for constructing several new macromolecular protein drugs for effective cell penetration.
authors.affiliation: Allan H Conney Laboratory for Anticancer Research, School of Chemical Engineering and Light Industry, Guangdong University of Technology, Guangzhou, China.
authors.forename: Weiping
authors.initials: W
authors.lastname: Lin
authors.affiliation: Allan H Conney Laboratory for Anticancer Research, School of Chemical Engineering and Light Industry, Guangdong University of Technology, Guangzhou, China; Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, New Jersey, USA.
authors.forename: Xi
authors.initials: X
authors.lastname: Zheng
authors.affiliation: Allan H Conney Laboratory for Anticancer Research, School of Chemical Engineering and Light Industry, Guangdong University of Technology, Guangzhou, China. Electronic address: huaqianwang@126.com.
authors.forename: Huaqian
authors.initials: H
authors.lastname: Wang
authors.affiliation: Allan H Conney Laboratory for Anticancer Research, School of Chemical Engineering and Light Industry, Guangdong University of Technology, Guangzhou, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Yu
authors.affiliation: Allan H Conney Laboratory for Anticancer Research, School of Chemical Engineering and Light Industry, Guangdong University of Technology, Guangzhou, China.
authors.forename: Xiaofen
authors.initials: X
authors.lastname: Zhou
authors.affiliation: Guangdong Entomological Institute, 510160 Guangzhou, China.
authors.forename: Yunxiao
authors.initials: Y
authors.lastname: Sun
authors.affiliation: Allan H Conney Laboratory for Anticancer Research, School of Chemical Engineering and Light Industry, Guangdong University of Technology, Guangzhou, China.
authors.forename: Suqing
authors.initials: S
authors.lastname: Zhao
authors.affiliation: Allan H Conney Laboratory for Anticancer Research, School of Chemical Engineering and Light Industry, Guangdong University of Technology, Guangzhou, China.
authors.forename: Zhiyun
authors.initials: Z
authors.lastname: Du
authors.affiliation: Allan H Conney Laboratory for Anticancer Research, School of Chemical Engineering and Light Industry, Guangdong University of Technology, Guangzhou, China; Wuyi University, Jiangmen, Guangdong, China.
authors.forename: Kun
authors.initials: K
authors.lastname: Zhang
mesh_terms: D045744:Cell Line, Tumor; D057846:Cell-Penetrating Peptides / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D002772:Cholera Toxin; D004337:Drug Carriers / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology
chemical_list: D057846:Cell-Penetrating Peptides; D004337:Drug Carriers; D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D002772:Cholera Toxin
keywords: Cholera toxin; Delivery carrier; EGFP; Protein complex; TAT