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Renaturation, purification, and characterization of human truncated macrophage colony-stimulating factor expressed in
K Yamanishi1, M Takahashi, T Nishida
1Cellular Technology Institute, Otsuka Pharmaceutical Co., Ltd., Tokushima.
Abstract:
A human truncated macrophage colony-stimulating factor (M-CSF) encoding the amino acid residues from 3 to 153 of the native M-CSF was expressed by using a two-cistron expression system in Escherichia coli. The truncated M-CSF found in inclusion bodies was renatured and had CSF activity. Purification, which included a QAE-ZeTa preparative cartridge concentration step followed sequentially by HPLC on TSK-gel Phenyl-5PW and TSK-gel DEAE-5PW columns, gave an overall yield of 63.8%. The purified truncated M-CSF had a specific activity of 4 x 10(7) units/mg of protein. Peptide mapping of a lysylendopeptidase digest by reversed-phase HPLC confirmed the amino acid sequence predicted from the cDNA sequence. SDS-PAGE of the purified truncated M-CSF gave a single band at 17 kDa under reducing conditions and at 32 kDa under non-reducing conditions. Activated Thiol-Sepharose 6B column chromatography and other experiments failed to detect any free cysteine residue in spite of the existence of 7 cysteine residues in the truncated M-CSF subunit. These results indicate that it is a dimeric structure linked by one or more intermolecular disulfide bonds.
Insights
This study successfully produced active truncated human macrophage colony-stimulating factor (M-CSF) in E. coli. The purified protein exhibits a dimeric structure linked by disulfide bonds, indicating its potential for further research.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Expression
Background:
- Macrophage colony-stimulating factor (M-CSF) is crucial for hematopoiesis.
- Production of functional M-CSF variants can be challenging.
- Truncated forms may offer specific biological properties or advantages in production.
Purpose of the Study:
- To express and purify a truncated human M-CSF (residues 3-153).
- To characterize the biological activity and structural properties of the recombinant protein.
- To confirm the integrity of the expressed protein sequence.
Main Methods:
- Two-cistron expression system in Escherichia coli.
- Inclusion body renaturation and purification using QAE-ZeTa, HPLC (TSK-gel Phenyl-5PW, DEAE-5PW).
- Peptide mapping, SDS-PAGE, and activated Thiol-Sepharose 6B chromatography.
Main Results:
- Truncated M-CSF was successfully expressed, renatured, and showed CSF activity.
- High-yield purification (63.8%) yielded protein with specific activity of 4 x 10(7) units/mg.
- SDS-PAGE indicated a dimeric structure (32 kDa non-reducing) linked by disulfide bonds, with no free cysteine residues detected.
Conclusions:
- The study established a method for producing active, truncated human M-CSF.
- The recombinant protein exists as a disulfide-bonded dimer, confirming its structural integrity.
- This work provides a foundation for exploring the functional roles of truncated M-CSF.