[Molecular design and construction of IL6D24-PE40KDEL, a novel recombinant interleukin6-pseudomonas exotoxin fusion protein, having targeted cytotoxicity for leukemias expressing interleukin6 receptors].
OBJECTIVE
A novel recombinant interleukin6-Pseudomonas exotoxin fusion protein, having targeted cytotoxicity for leukemic cells highly expressing IL6R has been molecularly designed and constructed in this study.


METHODS
Firstly, REDLK at C-terminus of Pseudomonas exotoxin PE40 was replaced with KDEL using point mutagenesis technology. Secondly, a cDNA encoding interleukin-6 devoid of N-terminal 24 amino acids [IL6D24] was fused to 5' terminus of PE40KDEL DNA by the method of gene splicing by overlap extension, which could generate recombinant IL6D24-PE40KDEL and IL6D24-Linker-PE40KDEL fusion genes. Thirdly, recombinant fusion genes IL6D24-PE40KDEL and IL6D24-Linker-PE40KDEL were ligated into the EcoR I and Sma I cloning sites in the pBV220 plasmids respectively and then transformed into E.coli HB101 cells. The expressed recombinant exotoxin fusion proteins were purified to electrophoresis purity by Mono Q column chromatography. Its selectively killing was tested by the MTS colorimetric method using both U937 and CEM cells lines.


RESULTS
Recombinant exotoxin fusion proteins IL6D24-PE40KDEL was expressed as a form of inclusion bodies at higher level of 40% approximately of total proteins in bacterial cells. Western blot showed that the purified products could react specifically with IL6 monoclonal antibody and PEA antiserum, respectively. IL6D24-PE40KDEL was selectively cytotoxic to U937 cells expressing IL6R-positive with ID50 of 250 ng/ml, and but not CEM cells expressing IL6R-negative.


CONCLUSIONS
Two novel recombinant interleukin6-Pseudomonas exotoxin fusion proteins IL6D24-PE40KDEL and IL6D24-Linker-PE40KDEL have been successfully constructed and they can selectively kill the leukemic cells expressing highly IL6R.
Zheng
Li-yan
LY
Department of Immunology, Affiliated Hospital for Academy of Military Medical Sciences, National Center of Biomedical Analysis Lab. of Immunoassay, Beijing 100039, China.
Xi
Yong-zhi
YZ
Kong
Fan-hua
FH
Cui
Jian-wu
JW
Liang
Fei
F
Liu
Nan
N
Sun
Yu-ying
YY
Guo
Si-qi
SQ
D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D000912:Antibodies, Neoplasm / Q000096:biosynthesis / Q000235:genetics; D000951:Antigens, Neoplasm / Q000096:biosynthesis / Q000276:immunology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D003602:Cytotoxicity, Immunologic; D004926:Escherichia coli / Q000235:genetics; D005098:Exotoxins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D015850:Interleukin-6 / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology*; D015458:Leukemia, T-Cell / Q000276:immunology* / Q000473:pathology; D010957:Plasmids / Q000235:genetics; D019947:Receptors, Interleukin-6 / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D020298:U937 Cells
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D001426:Bacterial Proteins; D005098:Exotoxins; C064195:IL-6-PE40 protein, chimeric; D015850:Interleukin-6; D019947:Receptors, Interleukin-6; D011993:Recombinant Fusion Proteins; C111749:pseudomonas exoprotein A protein, Pseudomonas aeruginosa; D036002:ADP Ribose Transferases