Expression and purification of recombinant human interleukin 4 in Escherichia coli / 生物工程学报
Chinese Journal of Biotechnology
; (12): 962-967, 2006.
Article
in Zh
| WPRIM
| ID: wpr-325442
Responsible library:
WPRO
ABSTRACT
Human interleukin 4 (IL-4) cDNA was optimized and synthesized according to E. coli preferred codon. A recombinant expression plasmid pET-30a (+)/rhIL-4 was constructed with the target cDNA inserted between Nde I and EcoR I sites, which can translate the mature IL-4 protein with an extra methionine residue at N-terminal. The expression vector was transformed into E. coli BL21 (DE3). The rhIL-4 protein was expressed in the inclusion body. By using the optimized fermentation conditions, the high expression level was achieved with the expression level as high as 35% of total protein obtained. A purification strategy has been designed which includes Q-Sepharose and SP-Sepharose ion-exchange chromatography and dialysis renaturation. The rhIL-4 was purified with the purity more than 98% and the yield of 40 mg per liter fermentation culture achieved. Western blot proved that the purified protein is IL-4. Amino acid sequencing revealed that N-terminal 16 residue sequence is identical to the theoretical sequence. Biological activity assay on TF-1 cells demonstrated that the rhIL-4 is active with an activity of 2.5 x 10(6) AU/mg. This study promises large scale production of rhIL-4.
Full text:
1
Index:
WPRIM
Main subject:
Recombinant Proteins
/
Molecular Sequence Data
/
Gene Expression
/
Chemistry
/
Blotting, Western
/
Amino Acid Sequence
/
Interleukin-4
/
Escherichia coli
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Fermentation
/
Genetics
Limits:
Humans
Language:
Zh
Journal:
Chinese Journal of Biotechnology
Year:
2006
Type:
Article