Enhanced and Secretory Expression of Human Granulocyte Colony Stimulating Factor by Bacillus subtilis SCK6

Abstract

This study describes a simplified approach for enhanced expression and secretion of a pharmaceutically important human cytokine, that is, granulocyte colony stimulating factor (GCSF), in the culture supernatant of Bacillus subtilis SCK6 cells. Codon optimized GCSF and pNWPH vector containing SpymwC signal sequence were amplified by prolonged overlap extension PCR to generate multimeric plasmid DNA, which was used directly to transform B. subtilis SCK6 supercompetent cells. Expression of GCSF was monitored in the culture supernatant for 120 hours. The highest expression, which corresponded to 17% of the total secretory protein, was observed at 72 hours of growth. Following ammonium sulphate precipitation, GCSF was purified to near homogeneity by fast protein liquid chromatography on a QFF anion exchange column. Circular dichroism spectroscopic analysis showed that the secondary structure contents of the purified GCSF are similar to the commercially available GCSF. Biological activity, as revealed by the regeneration of neutrophils in mice treated with ifosfamine, was also similar to the commercial preparation of GCSF. This, to our knowledge, is the first study that reports secretory expression of human GCSF in B. subtilis SCK6 with final recovery of up to 96 mg/L of the culture supernatant, without involvement of any chemical inducer.

Figure 1

Construction of the pNWPH-GCSF expression plasmid using prolonged overlap extension PCR/multimeric cloning strategy. Simple PCR generated 3′ and 5′ overhangs of insert (GCSF) and vector (pNWPH). These overhangs acted as primers during the formation of multimers. Circular plasmid pNWPH-GCSF was thereafter generated in B. subtilis by direct transformation of multimers containing GCSF gene. repB, replication protein B; Cat, chloramphenicol transferase gene; P_HbaII, promoter; SDgsiB, Shine-Dalgarno sequence of the gsiB gene; SPywmC, signal sequence.

Figure 2

(a) Restriction analysis of pNWPH-GCSF expression plasmid resolved on 1% agarose gel. M, 1 kb DNA size marker; Lane 1, undigested pNWPH-GCSF; Lane 2, pNWPH-GCSF after double digestion with NdeI and HindIII restriction endonucleases. (b) 13% SDS-PAGE analysis of TCA-acetone precipitated culture supernatant of transformed B. subtilis SCK6. Lane M represents protein size marker; Lanes 1–7, sample fractions collected at 24, 36, 48, 60, 72, 84, and 96 hours of cell growth. (c) Growth of recombinant B. subtilis harboring pNWPH-GCSF in 2x L-Mal medium. x-axis shows time in hours while primary y-axis reflects the concentration of GCSF (μg/mL) in culture supernatant, and secondary y-axis shows cell growth, monitored by absorbance measurement at 600 nm.

Figure 3

(a) Purification of recombinant human GCSF by FPLC on QFF column. Inset shows the purified GCSF eluted with 0.3 M NaCl concentration gradient. Blue and red colors show absorbance at A₂₈₀ and A₂₆₀, respectively. (b) CD spectrum of the recombinant in-house produced GCSF (solid line) and the commercially available GCSF preparation, that is, Filgrastim (dotted line), scanned over 185–260 nm range.

Figure 4

(a) GCSF biological activity assay. Left, mice being injected with GCSF by subcutaneous route; right, microscopic analysis of Giemsa stained slides wherein the encircled cells represent the neutrophil counts. (b) Mice in the sample and the control group received two different doses of GCSF (15 and 40 μg/mL/mouse). The control group was treated with 0.1% BSA in PBS. The abbreviations cGCSF and rhGCSF stand for commercially available GCSF and in-house produced recombinant human GCSF, respectively.