Mazurkiewicz-Pisarek Anna, Mazurkiewicz Alina, Mikiewicz Diana, Baran Piotr, Ciach Tomasz
Centre for Advanced Materials and Technologies, Warsaw University of Technology, Warszawa, Poland.
Science4Beauty LLC, Warszawa, Poland.
BioTechnologia (Pozn). 2023 Sep 25;104(3):247-262. doi: 10.5114/bta.2023.130728. eCollection 2023.
In this article, we have demonstrated the feasibility of generating an active form of recombinant blood coagulation factor VIII using an 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 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 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 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.
在本文中，我们证明了使用细菌表达系统生成活性形式的重组血液凝固因子VIII作为A型血友病潜在治疗方法的可行性。因子VIII（FVIII）是一种必需的血液凝固蛋白，是液相血液凝固系统的关键组成部分。到目前为止，所有可用的重组FVIII制剂都是使用真核表达系统生产的。哺乳动物细胞可以产生具有所有必要翻译后修饰的催化活性蛋白。然而，培养此类细胞既耗时又成本高昂，而且获得的产物量通常较低。与真核细胞不同，细菌培养成本低廉，并且能够在短时间内获得大量重组蛋白。通过本研究，我们旨在使用细菌表达系统获得重组血液凝固因子VIII，这是一种此前未用于此目的的方法。我们的研究包括血液凝固因子VIII的合成及其在原核系统中的表达。为实现这一目标，我们构建了一个包含合成因子VIII基因的原核表达载体，然后将其用于转化一种细菌菌株。通过质谱法确认了蛋白质表达，我们在确定最佳生长条件的同时评估了基因构建体的稳定性。该细菌菌株生产血液凝固因子VIII是在中试规模上进行的。我们确定了蛋白质的分离、变性和复性条件，随后证实了FVIII 的活性。
