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Cloning, soluble expression and purification of high yield recombinant hGMCSF in Escherichia coli.

Das KM, Banerjee S, Shekhar N, Damodaran K, Nair R, Somani S, Raiker VP, Jain S, Padmanabhan S - Int J Mol Sci (2011)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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.).

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.

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Related in: MedlinePlus

The biological activity of in-house hGMCSF was assessed using TF-1 cell proliferation assay. The activity data was analyzed statistically using Parallel Line Assay software (PLA 2.0). The doses are indicated on the horizontal axis, whereas the corresponding responses are represented on the vertical axis. The individual responses for each treatment are symbolized by the red squares for the standard preparation and by the blue circles for the sample preparation.
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f6-ijms-12-02064: The biological activity of in-house hGMCSF was assessed using TF-1 cell proliferation assay. The activity data was analyzed statistically using Parallel Line Assay software (PLA 2.0). The doses are indicated on the horizontal axis, whereas the corresponding responses are represented on the vertical axis. The individual responses for each treatment are symbolized by the red squares for the standard preparation and by the blue circles for the sample preparation.

Mentions: The TRX-GMCSF, containing a six His-tag in between the fusion partners TRX and GMCSF, was purified through Ni2+-NTA sepharose following the protocol described in the Experimental section. The purified fusion protein (Figure 3a, lane 2) after enterokinase cleavage and second round of purification yielded >95% pure hGMCSF protein (Figure 3a, lane 3) with a final yield of ∼44 mg/L and a fold purification of 2.5 (Table 1). Immunoblot analysis with mouse monoclonal anti-hGMCSF antibody confirmed the identity of the purified protein, which has a theoretical molecular mass 14.4 kDa (Figure 3b). The purity of the purified soluble GMCSF from the above fusion tag clone following the described method was analyzed by RP-HPLC and SE-HPLC for identity and similarity study with commercial GMCSF (Sigma, U.S.). RP-HPLC profiles of both soluble hGMCSF and commercial hGMCSF showed a similar pattern (Figure 4) at a retention time of 19.797 min with a purity of ∼95%, which is better than the commercial protein (∼90.2%), indicating the efficient separation and purification of the protein of interest. The commercial hGMCSF used was procured from Sigma (G 5035) and the product is supplied as a lyophilized powder from a 10 mM sodium citrate solution, pH 3.5, with no other proteinous material. This was also evident from the profile, with the absence of any major peak other than hGMCSF peak. The SE-HPLC analysis was carried out to determine the presence of GMCSF related impurities like aggregation and different conformational forms [17]. The chromatogram (Figure 5) shows that the in-house purified hGMCSF is ∼92% pure with no detectable aggregation or other conformational forms, while purity of the commercial GMCSF preparation was found to be relatively less (∼86.8%). The biological activity assay data indicate that the in-house hGMCSF is more active (potency 1.396) than the commercial preparation (Figure 6) and this could be partially attributed to the better purity of the in-house protein preparation.


Cloning, soluble expression and purification of high yield recombinant hGMCSF in Escherichia coli.

Das KM, Banerjee S, Shekhar N, Damodaran K, Nair R, Somani S, Raiker VP, Jain S, Padmanabhan S - Int J Mol Sci (2011)

The biological activity of in-house hGMCSF was assessed using TF-1 cell proliferation assay. The activity data was analyzed statistically using Parallel Line Assay software (PLA 2.0). The doses are indicated on the horizontal axis, whereas the corresponding responses are represented on the vertical axis. The individual responses for each treatment are symbolized by the red squares for the standard preparation and by the blue circles for the sample preparation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3111651&req=5

f6-ijms-12-02064: The biological activity of in-house hGMCSF was assessed using TF-1 cell proliferation assay. The activity data was analyzed statistically using Parallel Line Assay software (PLA 2.0). The doses are indicated on the horizontal axis, whereas the corresponding responses are represented on the vertical axis. The individual responses for each treatment are symbolized by the red squares for the standard preparation and by the blue circles for the sample preparation.
Mentions: The TRX-GMCSF, containing a six His-tag in between the fusion partners TRX and GMCSF, was purified through Ni2+-NTA sepharose following the protocol described in the Experimental section. The purified fusion protein (Figure 3a, lane 2) after enterokinase cleavage and second round of purification yielded >95% pure hGMCSF protein (Figure 3a, lane 3) with a final yield of ∼44 mg/L and a fold purification of 2.5 (Table 1). Immunoblot analysis with mouse monoclonal anti-hGMCSF antibody confirmed the identity of the purified protein, which has a theoretical molecular mass 14.4 kDa (Figure 3b). The purity of the purified soluble GMCSF from the above fusion tag clone following the described method was analyzed by RP-HPLC and SE-HPLC for identity and similarity study with commercial GMCSF (Sigma, U.S.). RP-HPLC profiles of both soluble hGMCSF and commercial hGMCSF showed a similar pattern (Figure 4) at a retention time of 19.797 min with a purity of ∼95%, which is better than the commercial protein (∼90.2%), indicating the efficient separation and purification of the protein of interest. The commercial hGMCSF used was procured from Sigma (G 5035) and the product is supplied as a lyophilized powder from a 10 mM sodium citrate solution, pH 3.5, with no other proteinous material. This was also evident from the profile, with the absence of any major peak other than hGMCSF peak. The SE-HPLC analysis was carried out to determine the presence of GMCSF related impurities like aggregation and different conformational forms [17]. The chromatogram (Figure 5) shows that the in-house purified hGMCSF is ∼92% pure with no detectable aggregation or other conformational forms, while purity of the commercial GMCSF preparation was found to be relatively less (∼86.8%). The biological activity assay data indicate that the in-house hGMCSF is more active (potency 1.396) than the commercial preparation (Figure 6) and this could be partially attributed to the better purity of the in-house protein preparation.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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.).

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.

Show MeSH
Related in: MedlinePlus