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Related Experiment Videos

Purification and partial characterization of recombinant human differentiation-stimulating factor.

C H Schmelzer1, L E Burton, C M Tamony

  • 1Department of Recovery Process Research and Development, Genentech, Inc., South San Francisco, California 94080.

Protein Expression and Purification
|September 1, 1990
PubMed
Summary

Recombinant human differentiation-stimulating factor (rhD-factor) was purified to over 95% and characterized. This glycoprotein demonstrated high stability and potent biological activity, even after deglycosylation.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Chemistry

Background:

  • Differentiation-stimulating factor (D-factor) plays a crucial role in cellular differentiation.
  • Production of recombinant human D-factor (rhD-factor) is essential for therapeutic and research applications.

Purpose of the Study:

  • To isolate and characterize recombinant human differentiation-stimulating factor (rhD-factor) produced in Chinese hamster ovary cells.
  • To assess the biological activity, stability, and glycosylation profile of rhD-factor.

Main Methods:

  • Isolation and purification of rhD-factor using chromatography techniques.
  • Molecular weight determination via SDS-PAGE and gel filtration.
  • Amino-terminal sequencing and glycosylation analysis.

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  • Biological activity assessment using ED50 determination.
  • Stability studies under various chemical and pH conditions.

    • Main Results:

    • rhD-factor was purified to >95% purity, exhibiting a molecular weight of approximately 45.6 kDa (SDS-PAGE) and 44.8 kDa (native conditions).
    • The protein is heavily glycosylated (30% neutral sugar, 12% sialic acid) and its amino-terminal sequence matched genomic predictions.
    • Deglycosylated rhD-factor retained comparable biological activity (ED50 = 0.40 ng/ml vs. 0.25 ng/ml for native).
    • rhD-factor demonstrated remarkable stability across a range of pH, denaturing agents (guanidine-HCl, SDS), and reducing conditions.

    Conclusions:

    • Purified rhD-factor is a stable, highly active glycoprotein suitable for further investigation.
    • Glycosylation significantly contributes to the protein's structure but is not essential for its biological activity.
    • The stability of rhD-factor under harsh conditions suggests potential for robust therapeutic formulations.