[High level secretory expression of recombinant human interleukin 18 in Pichia pastoris].
OBJECTIVE
To construct eukaryotic expression vector and express human interleukin 18 (hIL-18) in Pichia pastoris.


METHODS
The gene encoding of hIL-18 was amplification by PCR. The recombinant pPICZaC/hIL-18 was transformed into the Pichia pastoris X-33 strain via electroporation. The high level expression was selected and assayed by the methods of PCR, SDS-PAGE and Western blot. The rhIL-18 was purified by the methods of hydrophobic chromatography and anion exchange chromatography. The bioactivity of it was initially assayed.


RESULTS
The rhIL-18 was secreted into the supernatant and the concentration reached to 202 mg/L. The rhIL-18 was further identified by Western blot with specific antibody binding activity. The purity of the rhIl-18 reached about 95%. And rhIL-18 can synergistically induce PBMC to produce IFN-gamma with IL-2.


CONCLUSIONS
A rhIL-18 is successfully constructed and expressed in Pichia pastoris. And this contributes to further study of its function and activity.
Yang
Li-li
LL
Department of Immunology, Key Laboratory of Cancer Prevention and Therapy, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, China. yanglili2006@yahoo.com.cn
Wei
Feng
F
Liu
Hong
H
Li
Hui
H
Yu
Jin-pu
JP
Ren
Xiu-bao
XB
D015153:Blotting, Western; D002478:Cells, Cultured; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D020382:Interleukin-18 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D007376:Interleukin-2 / Q000494:pharmacology; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology
D020382:Interleukin-18; D007376:Interleukin-2; D011994:Recombinant Proteins; D007371:Interferon-gamma