[Expression, purification of tetanus toxin C fragment/cardiotrophin-1 recombinant fusion protein, target delivery to CNS and neurotrophy biology ability].
OBJECTIVE
Expression, purification of tetanus toxin C fragment/cardiotrophin-1 recombinant fusion protein (CT-1/TTC) in BL21 (DE3) E. coli, examined whether tetanus toxin C fragment mediate the cardiotrophin-1 target delivery to the central nervous system and the cardiotrophin-1 has the neurotrophic ability.


METHODS
Induction by IPTG, the fusion protein was expressed and then purified by GST affinity agarose. The interest protein was viewed by SDS-PAGE, further characterized by Western Blot Rat sciatic nerve transected model was selected. Using drug by nerve-regeneration-chamber and intramuscular injection. Execute these animals one week after the operation. The L4-L6 segments of the spinal cord were harvested after transaortic perfusion with 4% paraformaldehyde. The freeze sections of spinal tissues were stained with immunohistochemistry method. And select the new born SD rat sciatic nerve transected model, using CT-1/TTC fusion protein by muscle injection. Execute these animals one week after the operation. The L4-L6 segments of the spinal cord were harvested after transaortic perfusion with 4% paraformaldehyde. The freeze sections of spinal tissues were stained by Nissl's staining.


RESULTS
After induction, the fusion protein was about 15% of the total protein and the soluble part was predominant. Purified by GST fusion protein column, the interest protein's concentration is 2.7 g/L. The CT-1/TTC fusion protein was found in lumbar intumescentia by immunohistochemistry method. And after sciatic nerve transected, the numbers of cornu anterius medullae spinalis motoneurons in L4-L6 segments, compared to CT-1/TTC protein grope, have a lower survival rate.


CONCLUSIONS
The recombinant CT-1/TTC protein can be expressed and purified in BL21 (DE3) E. coli. This fusion protein has two biological activities of targeting delivery to central nervous system and protecting the cornu anterius medullae spinalis motoneurons.
Chen
Chao
C
Department of Orthopaedics, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China.
Wang
Jian
J
Yang
Ping
P
Zhou
Yue
Y
Zhang
Zheng-Feng
ZF
D000818:Animals; D000831:Animals, Newborn; D016207:Cytokines / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D004195:Disease Models, Animal; D004926:Escherichia coli / Q000378:metabolism; D005260:Female; D009046:Motor Neurons / Q000378:metabolism* / Q000473:pathology; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D012584:Sciatic Nerve / Q000293:injuries; D013116:Spinal Cord / Q000473:pathology; D013744:Tetanus Toxin / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*
D016207:Cytokines; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D013744:Tetanus Toxin; C040635:tetanus toxin fragment C; C092073:cardiotrophin 1