title: Recombinant expression and downstream processing of the disulfide-rich tumor-targeting peptide chlorotoxin.
abstract: Chlorotoxin (CTX) is a scorpion-derived disulfide-rich peptide that targets malignant tumors by binding the cell surface matrix metalloproteinase-2 and annexin A2. Various CTXs labeled with functional moieties have shown great potential for tumor diagnosis and treatment. In the present study, we established an efficient approach for preparing mature CTX that may be used for experimental and therapeutic purposes. The designed CTX precursors carried either a 6xHis-tag or a 6xHis-tag and a glutathione transferase (GST)-tag and were recombinantly expressed inEscherichia coli. Following S-sulfonation, the precursors were purified using immobilized metal-ion affinity chromatography. Subsequent to the removal of the tag by enterokinase cleavage andin vitrooxidative refolding, mature CTX was obtained with a considerable yield. The yield of mature CTX whose precursors carried a 6xHis-tag and a GST-tag (2 mg per liter of culture) was ∼10-fold that of the mature CTX whose precursors carried a 6xHis-tag (150-200μg per liter of culture). The folded CTX inhibited the migration of glioma cells in a concentration-dependent manner, suggesting it was biologically active.
authors.affiliation: Institute of Protein Research, College of Life Sciences and Technology, Tongji University, Shanghai 200092, P.R. China.
authors.forename: Xiao-Min
authors.initials: XM
authors.lastname: Wang
authors.forename: Xiao
authors.initials: X
authors.lastname: Luo
authors.forename: Zhan-Yun
authors.initials: ZY
authors.lastname: Guo
keywords: chlorotoxin; disulfides; gliomas; purification; recombinant expression; refolding