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.

Keywords: IMAC; TRX fusion; enterokinase; human granulocyte macrophage colony stimulating factor; on-column cleavage.

Figure 1.

Expression analysis of pET21a-hGMCSF and pET32a-hGMCSF. (a) Upper Panel: SDS-PAGE analysis of pET21a-hGMCSF expression in BL21 DE3 codon plus cells with IPTG induction, showing absence of visible expression. Lane M: molecular weight Marker; lane 1: Empty vector control; lane 2: expression from pET21-hGMCSF. Lower Panel: Immunoblot using mouse monoclonal anti-hGMCSF antibody showing low expression level of hGMCSF; (b) SDS-PAGE analysis of TRX-hGMCSF fusion protein expression. The pET32a-hGMCSF construct was transformed into BL21 (DE3) codon plus cells. The cells were induced with 1 mM IPTG for 3 h. Lane M: Molecular weight Marker; lane 1: Crude cell lysate; lane 2: Soluble fraction and lane 3: Insoluble fraction. Arrows indicate TRX-GMCSF protein.

Figure 2.

Expression of TRX-hGMCSF in 2 L fermenter in In-house medium. Various fermentation parameters were plotted against the batch hour. The cells were induced with 1 mM IPTG at an OD₆₀₀ of 18 and the culture was grown for another 3 h until it reached an OD₆₀₀ of 37.

Figure 3.

SDS-PAGE and Western blot analysis of purified hGMCSF. Soluble fraction containing TRX-hGMCSF was passed through Ni-NTA column and the fractions containing pure TRX-GMCSF were dialyzed and bound to Ni-NTA a second time in batch mode. The fusion protein was cleaved in the bound state with enterokinase enzyme in the presence of 1 mM CaCl₂. The flow through containing hGMCSF was collected and analyzed. (a) SDS-PAGE showing different purification steps. Lane M: Molecular weight Marker; lane 1: Soluble fraction containing TRX-hGMCSF fusion; lane 2: Purified TRX-GMCSF fusion, lane 3: purified hGMCSF; (b) Immunoblot using mouse monoclonal anti-hGMCSF antibody to confirm the identity of hGMCSF. Lane 1: Soluble fraction containing TRX-hGMCSF fusion; lane 2: Purified TRX-GMCSF fusion; lane 3: purified hGMCSF. Arrows represent the purified hGMCSF in both SDS-PAGE and immunoblot.

Figure 4.

Reverse phase chromatograph showing comparison of standard GMCSF and in-house purified hGMCSF with optimum mobile phase consisting of 0.1% TFA in 10% acetonitrile (a) and 0.1% TFA in 90% acetonitrile (b). Flow rate was maintained at 1 mL/min and detection was at 215 nm. Black line represents standard hGMCSF while red line indicates the in-house purified rhGMCSF.

Figure 5.

Size Exclusion HPLC profile of standard and in-house purified GMCSF preparations. Size exclusion chromatograph showing comparison of standard GMCSF and in-house purified hGMCSF with optimum mobile phase consisting of 1.15 g di-sodium hydrogen phosphate, 0.2 g of potassium hydrogen phosphate and 23.4 g of sodium chloride. Flow rate was maintained at 0.5 mL/min and detection was at 215 nm. Black line represents standard hGMCSF while red line indicates the in-house purified rhGMCSF.

Figure 6.

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.