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Preparative conversion of native human antithrombin to the latent form
1Plasma R&D, Octapharma AB, SE-112 75, Stockholm, Sweden. goran.karlsson@biovitrum.com
Protein Expression and Purification
|October 3, 2002
Summary
Researchers developed a method to convert native antithrombin (AT) into latent AT (L-AT). This latent form shows significant antiangiogenic and tumor-suppressing properties, offering potential therapeutic applications.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Native human antithrombin (AT) can be converted to a latent form (L-AT).
- Latent AT (L-AT) demonstrates potent antiangiogenic activity.
- L-AT has shown efficacy in suppressing tumor growth in preclinical models.
Purpose of the Study:
- To present a reliable method for inducing the conversion of native AT to L-AT.
- To characterize the resulting L-AT preparation.
Main Methods:
- Incubation of native AT at 60°C for 16 hours with 0.9 M ammonium sulfate in 5mM Hepes buffer (pH 7.4).
- Analysis of L-AT by affinity chromatography to quantify heparin affinity.
- Native polyacrylamide gel electrophoresis to assess for aggregates.
- Hydrophobic interaction chromatography for AT and L-AT separation.
Main Results:
- A method yielding over 70% recovery of L-AT was established.
- Affinity chromatography confirmed L-AT by identifying a low heparin affinity peak.
- Native PAGE demonstrated the absence of aggregates in the L-AT preparation.
- Hydrophobic interaction chromatography successfully separated AT from L-AT.
Conclusions:
- A robust method for generating L-AT from native AT has been developed.
- The characterized L-AT exhibits properties suitable for further investigation.
- This method facilitates the production of L-AT for potential therapeutic development in cancer and angiogenesis-related diseases.