We report for the first time that the C-terminal region of hG-CSF suffers from proteolytic degradation when human granulocyte colony-stimulating factor (hG-CSF) is directly expressed in Escherichia coli BL21(DE3). It is believed that the rapid proteolysis occurs at the C-terminus of hG-CSF that is very easily exposed to E. coli protease(s) during a short period following protein synthesis and prior to completion of the formation of the inclusion body. The recombinant hG-CSF that is expressed with an N-terminal fusion partner is effectively protected from the proteolysis. It seems that since the N-terminus of hG-CSF is located very close to the C-terminus, the presence of the N-terminal fusion partner masks the C-terminal region of hG-CSF and protects it from proteolytic degradation by E. coli protease(s). Furthermore, the solubility of hG-CSF markedly increased in E. coli cytoplasm when a stress-responsive and aggregation-resistant protein, i.e. aspartate carbamoyl-transferase catalytic chain (PyrB) was used as a novel N-terminal fusion partner proteins.
Bibliographical noteFunding Information:
We thank Professor Hang Chul Shin at Soongsil University for kindly providing the gene clones of hG-CSF. This study was supported by the National Research Laboratory Project of the Ministry of Education, Science and Technology (grant no. ROA-2007-000-20084-0) and by the Korea Health 21 R&D Project of the Ministry of Health & Welfare of the Republic of Korea (grant no. A050750). This work was also supported by the Second Brain Korea 21 Project. Additional support from Korea Research Foundation (grant no. KRF-2004-005-D00057) are also appreciated.
- C-terminal proteolysis
- E. coli BL21(DE3)
- Human granulocyte colony-stimulating factor (hG-CSF)
- N-terminal fusion partner
ASJC Scopus subject areas
- Applied Microbiology and Biotechnology