重组蛋白的生产和纯化对于获得纯MPT64蛋白至关重要。由于蛋白质表位在纯化过程中可能会发生构象变化,这项研究,因此,研究了一种有效的快速纯化方法,以产生高度细胞内纯的MPT64蛋白,而不会在变性条件下引起表位的构象变化。从大肠杆菌中分离MPT64并使用凝胶SDS-PAGE进行电泳。然后,用电洗脱和被动洗脱两种方法切除和纯化所需的蛋白带。使用MALDI-TOFMS通过肽质量指纹鉴定鉴定分离的蛋白质并与IgG抗MPT64反应,并且使用Western印迹确认分离的蛋白质与IgY抗MPT64的交叉反应性。结果表明,这两种方法都产生了纯MPT64蛋白,并且基于MALDI-TOFMS结果确认MPT64蛋白。这两种方法均未导致MPT64蛋白中的表位改变,因此其可与两种抗体特异性反应。电洗脱(2030±41µg/mL)的MPT64蛋白产量高于被动洗脱(179.5±7.5µg/mL)。因此,由此可以推断,电洗脱法是比被动洗脱法更有效的纯化MPT64蛋白并维持其表位的方法。
The production and purification of recombinant proteins are crucial to acquiring pure MPT64 protein. Due to the fact that protein epitopes may undergo conformational changes during purification, this study, therefore, investigated an effective rapid purification method to produce highly intracellular pure MPT64 protein without causing conformational changes in the epitope under denaturing conditions. MPT64 was isolated from E. coli and electrophoresed using gel SDS-PAGE. Then, the desired protein bands were excised and purified with two methods:
electroelution and passive elution. The isolated protein was identified via peptide mass fingerprinting using MALDI-TOF MS and reacted with IgG anti-MPT64, and the cross-reactivity of the isolated protein with IgY anti-MPT64 was confirmed using Western blot. The results show that both of these methods produced pure MPT64 protein, and the MPT64 protein was confirmed based on the MALDI-TOF MS results. Neither of these two methods resulted in epitope changes in the MPT64 protein so it could react specifically with both antibodies. The yield of MPT64 protein was higher with
electroelution (2030 ± 41 µg/mL) than with passive elution (179.5 ± 7.5 µg/mL). Thus, it can be inferred that the
electroelution method is a more effective method of purifying MPT64 protein and maintaining its epitope than the passive elution method.