Cloning, expression, purification, distribution and kinetics characterization of the bacterial beta-galactosidase fused to the cytoplasmic transduction peptide in vitro and in vivo.
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-beta-gal in vitro and (2) examine the kinetics and tissue distribution of the CTP-beta-gal fusion protein in mice. A CTP-beta-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 microg 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. beta-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 24h 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.
Huang
Shi-Feng
SF
Department of Clinical Hematology, Key Laboratory of Laboratory Medical Diagnostics Designated by the Ministry of Education, Faculty of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, PR China.
Liu
Ding-Bin
DB
Zeng
Jian-Ming
JM
Yuan
Ying
Y
Xiao
Qing
Q
Sun
Cheng-Ming
CM
Li
Chun-Li
CL
Tao
Kun
K
Wen
Jian-Ping
JP
Huang
Zong-Gan
ZG
Feng
Wen-Li
WL
D000818:Animals; D001426:Bacterial Proteins / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002461:Cell Line, Transformed; D003593:Cytoplasm / Q000378:metabolism*; D016503:Drug Delivery Systems; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D007150:Immunohistochemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014018:Tissue Distribution; D001616:beta-Galactosidase / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase