Expression, purification, and characterization of recombinant human interleukin 24 in Escherichia coli.
Interleukin-24 (IL-24) can induce apoptosis of a broad range of tumor cells, and this function of IL-24 is independent of classic tumor suppressor genes, such as p53, Rb and p16. Here, we report the expression, purification and preparation of a recombinant IL-24 protein (rIL-24) without post-translational modifications, which may selectively induce apoptosis of tumor cells in vitro. We found that non-fusion rIL-24 was not able to be expressed by vectors pET11c, 28a, and 22b in Escherichia coli. To obtain recombinant non-fusion IL-24 protein, the encoding region for IL-24 was cloned between KpnI and BamHI in pET32a. The Trx (Thioredoxin)/IL-24 fusion proteins were expressed in the form of inclusion bodies in E. coli host strain BL21 (DE21). The expression level was more than 30% of total cell lysate. Inclusion bodies were disrupted, washed, and isolated at pH 9.0, and were completely dissolved in a buffer containing 2M urea at pH 9.0. After nickel ion metal affinity chromatography, gel filtration chromatography, and renaturation, the refolded fusion proteins with a purity of >96% were obtained. Trx/IL-24 proteins were digested by enterokinase (EK) to both Trx and rIL-24 fragments which then were separated by cation exchange chromatography. Cell proliferation experiments proved that the rIL-24 (98% purity) retains its cancer-selective apoptosis-inducing properties. This result suggested that the rIL-24 may have cancer therapeutic applications.
Yang
Jun
J
Department of Clinical Microbiology and Immunology, Faculty of Medical Laboratory Science, Third Military Medical University, 30 Sha Ping Ba Gaotanyan Street, Chongqing 400038, PR China. w8301991@yahoo.com
Zhang
Weijun
W
Liu
Kaiyun
K
Jing
Shenrong
S
Guo
Gang
G
Luo
Ping
P
Zou
Quanming
Q
D001483:Base Sequence; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D007378:Interleukins / Q000235:genetics* / Q000302:isolation & purification* / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D000099180:Interleukin-24
D017931:DNA Primers; D007378:Interleukins; D011993:Recombinant Fusion Proteins; D000099180:Interleukin-24