Construction of intein-mediated hGMCSF expression vector and its purification in Pichia pastoris.
As a novel attempt for the intracellular recombinant protein over expression and easy purification from Pichia pastoris, the therapeutic cytokine human granulocyte macrophage colony stimulating factor (hGMCSF) gene was fused to an intein-chitin-binding domain (gene from pTYB11 vector) fusion tag by overlap extension PCR and inserted into pPICZB vector, allowing for the purification of a native recombinant protein without the need for enzymatic cleavage. The fusion protein under the AOX1 promoter was integrated into the P. pastoris genome (SMD 1168) and the recombinant Pichia clones were screened for multicopy integrants. Expression of hGMCSF was done using glycerol and methanol based synthetic medium by three stage cultivation in a bioreactor. Purification of the expressed hGMCSF fusion protein was done after cell disruption and binding of the solubilized fusion protein to chitin affinity column, followed by DTT induced on column cleavage of hGMCSF from the intein tag. In this study, final biomass of 89 g dry cell weight/l and purified hGMCSF of 120 mg/l having a specific activity of 0.657 x 10(7) IU/mg was obtained. This strategy has an edge over the other--His or--GST based fusion protein purification where non-specific protein binding, expensive enzymatic cleavage and further purification of the enzyme is required. It distinguishes itself from all other purification systems by its ability to purify, in a single chromatographic step.
Srinivasa Babu
K
K
Centre for Biotechnology, Anna University, Chennai 600025, India.
Antony
Aju
A
Muthukumaran
T
T
Meenakshisundaram
S
S
D015153:Blotting, Western; D002460:Cell Line; D005285:Fermentation; D005822:Genetic Vectors; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000302:isolation & purification* / Q000378:metabolism*; D006801:Humans; D047668:Inteins; D007700:Kinetics; D010843:Pichia / Q000254:growth & development / Q000378:metabolism*; D013816:Thermodynamics
D016178:Granulocyte-Macrophage Colony-Stimulating Factor