Expression, purification, and characterization of a functional mutant recombinant human interleukin-2.
In the current study, a mutant recombinant human interleukin-2 (MhIL-2) was generated using site-directed mutagenesis. The bacteria transformed with plasmid pET15b-MhIL-2 were cultured in LB medium containing 0.6mM IPTG for 8 hours at 27°C. Approximately 90% of His-MhIL-2 was efficiently expressed in soluble form. Purification efficiency was optimized using a number of strategies, including nickel ion chelating chromatography, desalting chromatography, thrombin cleavage and Superdex 75 gel filtration chromatography. The final product had >95% purity. PBMCs, CD4+ and CD8+ T cell proliferation assays revealed that one such mutant has identical functional property to the wild-type hIL-2. In summary, we generated a mutant hIL-2 that is functionally identical to wild-type hIL-2.
Clinical Immunologic Laboratory, Affiliated Hospital of Medical College Qingdao University, No.16 Jiangsu Road, Qingdao 266003, China. jocklmj@yahoo.com.cn
Mingjun
M
Liu
Bin
B
Wang
Guirong
G
Sun
Dongmeng
D
Qian
Zhiyong
Z
Yan
Xuxia
X
Song
Shouyi
S
Ding
D015153:Blotting, Western; D018414:CD8-Positive T-Lymphocytes / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology
D007376:Interleukin-2; D011993:Recombinant Fusion Proteins