Single step intein-mediated purification of hGMCSF expressed in salt-inducible E. coli.
Human granulocyte-macrophage colony stimulating factor (hGMCSF) is an important therapeutic cytokine. As a novel attempt to purify hGMCSF protein, without the enzymatic cleavage of the affinity tag, an intein-based system was used. The gene was fused by overlap extension PCR to the intein sequence at its N-terminal in pTYB11 vector. The hGMCSF was expressed as a fusion protein in E. coli BL21(DE3), and E. coli GJ1158. In the former, the protein was expressed as inclusion bodies and upon purification the yield was 7 mg/l with a specific activity of 0.5 x 10(7) IU/mg. In salt-inducible E. coli GJ1158, hGMCSF was expressed in a soluble form at 20 mg/l and a specific activity of 0.9 x 10(7) IU/mg. The intein-hGMCSF was purified on a chitin affinity column by cleaving intein with 50 mM DTT resulting in a highly pure 14.7 kDa hGMCSF.
Srinivasa Babu
K
K
Centre for Biotechnology, Anna University, Chennai, 600 025, India.
Muthukumaran
T
T
Antony
Aju
A
Prem Singh Samuel
S D
SD
Balamurali
M
M
Murugan
V
V
Meenakshisundaram
S
S
D002846:Chromatography, Affinity / Q000379:methods; D004229:Dithiothreitol / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D047668:Inteins / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification*
D011993:Recombinant Fusion Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D004229:Dithiothreitol