title: Cloning, soluble expression and purification of high yield recombinant hGMCSF in Escherichia coli.
abstract: Expression of human granulocyte macrophage colony stimulating factor (hGMCSF), a cytokine of therapeutic importance, as a thioredoxin (TRX) fusion has been investigated in Escherichia coli BL21 (DE3) codon plus cells. The expression of this protein was low when cloned under the T7 promoter without any fusion tags. High yield of GMCSF was achieved (∼88 mg/L of fermentation broth) in the shake flask when the gene was fused to the E. coli TRX gene. The protein was purified using a single step Ni(2+)-NTA affinity chromatography and the column bound fusion tag was removed by on-column cleavage with enterokinase. The recombinant hGMCSF was expressed as a soluble and biologically active protein in E. coli, and upon purification, the final yield was ∼44 mg/L in shake flask with a specific activity of 2.3 × 10(8) U/mg. The results of Western blot and RP-HPLC analyses, along with biological activity using the TF-1 cell line, established the identity of the purified hGMCSF. In this paper, we report the highest yield of hGMCSF expressed in E. coli. The bioreactor study shows that the yield of hGMCSF could be easily scalable with a yield of ∼400 mg/L, opening up new opportunities for large scale production hGMCSF in E. coli.
authors.affiliation: Clone Development Team, Lupin Limited, Biotechnology R & D, Gat #1156, Ghotawade Village, Mulshi Taluka, Pune-411042, India; E-Mails: krishnap@lupinpharma.com (K.M.P.D.); sampalibanerjee@lupinpharma.com (S.B.).
authors.forename: Krishna M P
authors.initials: KM
authors.lastname: Das
authors.forename: Sampali
authors.initials: S
authors.lastname: Banerjee
authors.forename: Nivedita
authors.initials: N
authors.lastname: Shekhar
authors.forename: Karpagavalli
authors.initials: K
authors.lastname: Damodaran
authors.forename: Rahul
authors.initials: R
authors.lastname: Nair
authors.forename: Sandeep
authors.initials: S
authors.lastname: Somani
authors.forename: Veena P
authors.initials: VP
authors.lastname: Raiker
authors.forename: Shweta
authors.initials: S
authors.lastname: Jain
authors.forename: Sriram
authors.initials: S
authors.lastname: Padmanabhan
mesh_terms: D061249:Batch Cell Culture Techniques; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D013879:Thioredoxins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor