Cloning, expression, purification, distribution and kinetics characterization of the bacterial β-galactosidase fused to the cytoplasmic transduction peptide in vitro and in vivo

Abstract Cytoplasmic transduction peptide (CTP) offers exciting therapeutic opportunities for the treatment of many diseases caused by cytoplasmic functional molecules. It can transduce large, biologically active proteins into the cytoplasmic compartment of several mammalian cells. However, other intriguing features of CTP, including its activity in vitro , and distribution and tissue infiltration abilities in vivo , remain to be explored. The present study was initiated to (1) further confirm the cytoplasmic localization preference and the enzymatic activity of the transduced CTP-β-gal in vitro and (2) examine the kinetics and tissue distribution of the CTP-β-gal fusion protein in mice. A CTP-β-gal fusion protein was expressed in Escherichia coli and either transduced into BaF3-BCR/ABL cells or administered intravenously into female Balb/C mice at a dose of 100 μg per mouse. Its localization in BaF3-BCR/ABL cells was evaluated by immunocytochemistry and in situ X-gal staining, and its distribution in various tissues was analyzed both by in situ X-gal staining and quantitative enzymatic activity assay. β-Galactosidase enzyme activity was observed in BaF3-BCR/ABL cells and in all tissues tested, with peak activity occurring at 15 min in most tissues and at 24 h in brain. These data will not only allow rational selection of delivery schedules for therapeutic CTP, but will also aid the use of CTP fusion protein transduction in the development of protein therapeutics targeting the cytoplasmic compartment both in vitro and in vivo .