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

نویسندگان

  • Shaista Bashir
  • Saima Sadaf
  • Sajjad Ahmad
  • Muhammad Waheed Akhtar
چکیده

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.

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

منابع مشابه

Optimization of secretory expression of recombinant hGM-CSF in high cell density cultivation of recombinant Escherichia coli using Taguchi statistical method

Human granulocyte macrophage colony stimulating factor (hGM-CSF) has many therapeutic applications.In this study, in order to verify the purification process, the effect of carbon source, IPTG concentration andpost-induction time on the secretion of recombinant hGM-CSF into the culture medium by recombinantEscherichia coli during high cell density cultivation were evaluated by...

متن کامل

Site-Directed Mutagenesis in Human Granulocyte-colony Stimulating Factor, Cloning and Expression in Escherichia coli

Human granulocyte colony stimulating factor (hG-CSF) induces proliferation and differentiation of granulocyte progenitor cells. This glycoprotein is currently being used for treatment of neutropenia, in patients who have undergone bone marrow transplantation. So far, different researchers have tried to enhance hG-CSF biological activity and stability. In this study, Polymerase Chain Reaction (P...

متن کامل

Expression of a Chimeric Protein Containing the Catalytic Domain of Shiga-Like Toxin and Human Granulocyte Macrophage Colony-Stimulating Factor (hGM-CSF) in Escherichia coli and Its Recognition by Reciprocal Antibodies

Fusion of two genes at DNA level produces a single protein, known as a chimeric protein. Immunotoxins are chimeric proteins composed of specific cell targeting and cell killing moieties. Bacterial or plant toxins are commonly used as the killing moieties of the chimeric immunotoxins. In this investigation, the catalytic domain of Shiga-like toxin (A1) was fused to human granulocyte macrophage ...

متن کامل

The Expression of Human Granulocyte Macrophage Colony Stimulating Factor by Heat-Induction in Escherichia coli

A self-regulated high-copy number plasmid containing chloramphenicol resistant gene, for the production of recombinant proteins under the regulation of bacteriophage ?pL promoter, was constructed. The designed 5024 base pair expression plasmid contained a heat sensitive repressor cI857 coding gene to regulate the function of ?pL promoter under heat shock induction. Using the constructed vector,...

متن کامل

ذخیره در منابع من


  با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

عنوان ژورنال:

دوره 2015  شماره 

صفحات  -

تاریخ انتشار 2015