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High level expression of active human prothrombin in a vaccinia virus expression system
F G Falkner1, P L Turecek, R T MacGillivray
1Immuno AG, Biomedical Research Center, Orth/Donau, Austria.
Thrombosis and Haemostasis
|August 3, 1992
Summary
Researchers developed an efficient method for expressing recombinant human prothrombin using the vaccinia virus system in mammalian cells. This technique offers a viable alternative for producing human prothrombin, aiding further research into its structure and function.
Area of Science:
- Biotechnology
- Molecular Biology
- Protein Expression
Background:
- Human prothrombin (Factor II) is a crucial protein in the blood coagulation cascade.
- Efficient expression systems are needed for studying prothrombin's structure-function relationships and developing therapeutics.
Purpose of the Study:
- To establish an efficient and time-saving procedure for expressing recombinant human prothrombin.
- To evaluate the vaccinia virus expression system in various mammalian cell lines for prothrombin secretion.
Main Methods:
- Utilized the vaccinia virus expression system to express recombinant human prothrombin.
- Tested expression levels in kidney cell lines (Vero, BHK) and a human cell line (Hela).
- Quantified prothrombin expression levels in micrograms and milliunits per cell per day.
Main Results:
- Vero, BHK, and Hela cell lines efficiently secreted human prothrombin.
- Achieved expression levels of 3-4 µg of Factor II per 10^6 cells/day (18-23 mU/10^6 cells/day).
- Expression levels were comparable to those from amplified transformed Chinese Hamster Ovary (CHO) cells.
Conclusions:
- The vaccinia virus/Vero cell system provides an efficient alternative for recombinant human prothrombin expression.
- This system facilitates further elucidation of prothrombin structure-function relationships and interactions with effectors.