High-Level Secretory Expression and Purification of Recombinant Human Interleukin 1 Beta in Pichia pastoris.
As an important pro-inflammatory cytokine, interleukin-1beta (IL-1β) participates in a variety of physiological and pathological responses. In order to obtain higher yielded recombinant human interleukin-1 beta (rhIL-1β), we cloned hIL-1β cDNA sequences based on the coding sequence of human mature IL-1β. After recombinant pPICZαA/hIL-1β was separated and sequenced, we transformed recombinant pPICZαA/hIL-1β into Pichia pastoris GS115, SMD1168 and X-33 strain via electroporation. The results showed that recombinant pPICZαA/ hIL-1β had the highest expression level in X-33 Pichia pastoris. Subsequently, rhIL-1β was purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and identified by Western blot. Then the fermentation process was optimized to increase product yield. Under the fermentation conditions of the absorption value of fermentation liquor before induction of 600, oxygen concentration of 20%, methanol concentration of 0.25% with pH 5.0, the yield of rhIL-1β reached to 250 mg/L after 72 h induction at 26°C. After aqueous two-phase extraction combined with chromatography, the purity of rhIL-1β was 95% and the yield was up to 85%. The biological activity of rhIL-1β was detected by MTT assay, and the result showed that rhIL-1β significantly inhibited the growth and proliferation of B16 melanoma cells.
Pengfei
P
Li
Ganggang
G
Yang
Xiaofang
X
Geng
Jinbao
J
Shi
Bin
B
Li
Ze
Z
Wang
Quanyi
Q
Zhang
Yajuan
Y
Yang
State Key Laboratory Cultivation Base for Cell Differentiation Regulation, Henan Normal University, Xinxiang 453007, China. cellkeylab@126.com.
Cunshuan
C
Xu
D002460:Cell Line; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D006801:Humans; D053583:Interleukin-1beta / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D010843:Pichia / Q000235:genetics* / Q000254:growth & development; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
C508585:IL1B protein, human; D053583:Interleukin-1beta; D011994:Recombinant Proteins