Large-scale purification and characterization of recombinant human stem cell factor in Escherichia coli.
The pharmacological importance of recombinant human stem cell factor (rhSCF) has increased the demand to establish effective and large-scale production and purification processes. A good source of bioactive recombinant protein with capability of being scaled-up without losing activity has always been a challenge. The objectives of the study were the rapid and efficient pilot-scale expression and purification of rhSCF. The gene encoding stem cell factor (SCF) was cloned into pBV220 and transformed into Escherichia coli. The recombinant SCF was expressed and isolated using a procedure consisting of isolation of inclusion bodies (IBs), denaturation, and refolding followed by chromatographic steps toward purification. The yield of rhSCF reached 835.6 g/20 L, and the expression levels of rhSCF were about 33.9% of the total E. coli protein content. rhSCF was purified by isolation of IBs, denaturation, and refolding, followed by SP-Sepharose chromatography, Source 30 reversed-phase chromatography, and Q-Sepharose chromatography. This procedure was developed to isolate 5.5 g of rhSCF (99.5% purity) with specific activity at 0.96 × 106IU/mg, endotoxin levels of pyrogen at 1.0 EU/mg, and bacterial DNA at 10 ng/mg. Pilot-scale fermentations and purifications were set up for the production of rhSCF that can be upscaled for industry.
Institute of Ecological Forestry, Faculty of Forestry, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.
Liang-Hua
LH
Chen
College of Life Sciences, Sichuan University, Chengdu, People's Republic of China.
Feng
F
Cai
Institute of Ecological Forestry, Faculty of Forestry, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.
Dan-Ju
DJ
Zhang
Institute of Ecological Forestry, Faculty of Forestry, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.
Li
L
Zhang
Institute of Ecological Forestry, Faculty of Forestry, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.
Peng
P
Zhu
Institute of Ecological Forestry, Faculty of Forestry, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.|State Key Laboratory of Hydraulics and Mountain River Engineering, Sichuan University, Chengdu, People's Republic of China.
Shun
S
Gao
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005285:Fermentation; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D019089:Stem Cell Factor / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D011994:Recombinant Proteins; D019089:Stem Cell Factor
fermentation; inclusion bodies; pilot scale; purification process; refolding