Expression and purification of recombinant human interleukin 4 in Escherichia coli.
- Author:
Yan QIU
1
;
Jiu-Ru SUN
;
Yang-Bin HUANG
;
Zhi-Hua HUANG
;
Lin-Jie CHEN
;
Yong LIU
;
Yue-Xin LIN
Author Information
1. College of Life Science, Fujian Normal University, Fuzhou 350007, China. qybio@163.com
- Publication Type:Journal Article
- MeSH:
Amino Acid Sequence;
Blotting, Western;
Escherichia coli;
genetics;
metabolism;
Fermentation;
Gene Expression;
Humans;
Interleukin-4;
biosynthesis;
chemistry;
isolation & purification;
metabolism;
Molecular Sequence Data;
Recombinant Proteins;
biosynthesis;
chemistry;
isolation & purification;
metabolism
- From:
Chinese Journal of Biotechnology
2006;22(6):962-967
- CountryChina
- Language:Chinese
-
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.
