Expression, purification, and functional characterization of recombinant human interleukin-7.
Human interleukin-7 (IL-7) is a member of the interleukin family. Numerous studies have demonstrated IL-7's effect on B- and T-cell development as well as its potential in various clinical applications. Previously, a study reported that IL-7 could be purified from inclusion bodies using a prokaryotic system, however, the required refolding step limits the recovery rate. This study was designed to produce a bioactive recombinant human IL-7 (rhIL-7) in a eukaryotic expression system in order to obtain higher yields of the protein with simpler purification steps. We cloned human IL-7 cDNA and successfully expressed active recombinant protein in yeast using the Pichia pastoris expression system. A simple purification strategy was established to purify the rhIL-7 from the fermentation supernatant, yielding 35 mg/L at 95% purity by the use of a common SP Sepharose FF cation-exchange chromatography. Functional analysis of the purified rhIL-7 by the pre-B cell MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) proliferation assay demonstrated a specific activity comparable to commercial sources. These results suggest that purification of rhIL-7 from yeast provides a sound strategy for large-scale production of the rhIL-7 for clinical applications as well as basic researches.
Luo
Yong
Y
Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Science Park, Guangzhou 510663, China.
Kong
Xiangping
X
Xu
Aimin
A
Jin
Shouguang
S
Wu
Donghai
D
D019149:Bioreactors; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D005285:Fermentation; D006801:Humans; D015851:Interleukin-7 / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D012685:Sepharose; D013778:Tetrazolium Salts; D013844:Thiazoles; D014170:Transformation, Genetic
C508605:IL7 protein, human; D015851:Interleukin-7; D011994:Recombinant Proteins; D013778:Tetrazolium Salts; D013844:Thiazoles; C097574:sepharose fast flow SP; D012685:Sepharose; C022616:thiazolyl blue