[Construction and expression of protein self-splicing prokaryotic expression vector pTWIN1- AcAPc2].
To express recombinant Ancylostoma caninum anticoagulant peptide-c2 (AcAPc2), a whole cDNA fragment encoding AcAPc2 was achieved by ligation- PCR and inserted into prokaryotic expression vector pTWIN1 for constructing the specific self-splicing prokaryotic expression vector, pTWIN1-AcAPc2; positive recombinants were transformed into E. coli ER2566 for expression research. The recombinant protein, AcAPc2-intein2-CBD, was soluble and expressed in E. coli ER2566 (about 30.1% fusion protein in total protein). AcAPc2-intein2-CBD was characterized to be 41 KD by SDS-PAGE and identified by Western-blot. The recombinant fusion protein was purified to a efficiently high degree by chitin affinity chromatography. After the process of specific self-splicing induced by beta-Mercaptoethanol, the target protein, AcAPc2, was obtained, characterized to be 21 KD by SDS-PAGE and migrated as a dimmer. Molecular weight of AcAPc2 conformed to native dimmer. Bio-information analysis indicated relationship between secondary construction of AcAPc2 and biologic function. These findings greatly facilitate the purification of AcAPc2 and are very important for the additional studies on its anti-coagulation mechanism and its clinical application as anti-coagulation medicine.
Yang
Bo
B
Department of Biochemistry and Molecular Biology, Basic and Forensic Medical College, Sichuan University, Chengdu 610041, China.
Chen
Shouchun
S
Tong
Yu
Y
Qin
Yang
Y
D000818:Animals; D004285:Dogs; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D017238:Genes, Helminth; D005822:Genetic Vectors; D015801:Helminth Proteins / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011387:Prokaryotic Cells / Q000378:metabolism; D012326:RNA Splicing; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000494:pharmacology
D015801:Helminth Proteins; D011993:Recombinant Fusion Proteins; C094339:anticoagulant protein, Ancylostoma caninum