Current issue
Archive
About the journal
Editorial board
Abstracting and indexing
Subscription
Contact
Instructions for authors
Publication charge
Ethical standards and procedures
Editorial System
Submit your Manuscript
|
3/2023
vol. 104 abstract:
RESEARCH PAPERS
Expression of the gene encoding blood coagulation factor VIII without domain B in E. coli bacterial expression system
Anna Mazurkiewicz-Pisarek
1
,
Alina Mazurkiewicz
1
,
Diana Mikiewicz
2
,
Piotr Baran
3
,
Tomasz Ciach
4
BioTechnologia vol. 104(3) ∙ pp. 247–262 ∙ 2023
Online publish date: 2023/09/25
View
full text
Get citation
ENW EndNote
BIB JabRef, Mendeley
RIS Papers, Reference Manager, RefWorks, Zotero
AMA
APA
Chicago
Harvard
MLA
Vancouver
In this article, we have demonstrated the feasibility of generating an active form of recombinant blood coagulation factor VIII using an E. coli bacterial expression system as a potential treatment for hemophilia type A. Factor VIII (FVIII), an essential blood coagulation protein, is a key component of the fluid phase blood coagulation system. So far, all available recombinant FVIII formulations have been produced using eukaryotic expression systems. Mammalian cells can produce catalytically active proteins with all the necessary posttranslational modifications. However, cultivating such cells is time-consuming and highly expensive, and the amount of the obtained product is usually low. In contrast to eukaryotic cells, bacterial culture is inexpensive and allows the acquisition of large quantities of recombinant proteins in a short time. With this study, we aimed to obtain recombinant blood coagulation factor VIII using the E. coli bacterial expression system, a method not previously explored for this purpose. Our research encompasses the synthesis of blood coagulation factor VIII and its expression in a prokaryotic system. To achieve this, we constructed a prokaryotic expression vector containing a synthetic factor VIII gene, which was then used for the transformation of an E. coli bacterial strain. The protein expression was confirmed by mass spectrometry, and we assessed the stability of the gene construct while determining the optimal growth conditions. The production of blood coagulation factor VIII by the E. coli bacterial strain was carried out on a quarter-technical scale. We established the conditions for isolation, denaturation, and renaturation of the protein, and subsequently confirmed the activity of FVIII.
keywords:
factor VIII, hemophilia type A, recombinant coagulation factor VIII, prokaryotic expression system, E coli, recombinant protein production system |