Nye S H, Pelfrey C M, Burkwit J J, Voskuhl R R, Lenardo M J, Mueller J P
Alexion Pharmaceuticals, Inc., New Haven, CT 06511, USA.
Mol Immunol. 1995 Oct;32(14-15):1131-41. doi: 10.1016/0161-5890(95)00066-6.
We have designed and expressed in bacteria a recombinant fetal form of human myelin basic protein (21.5 kDa isoform; rhMBP21.5), a candidate autoantigen in multiple sclerosis. An exon 2 insertion, carboxy-terminal histidine tag and preferred bacterial codons differentiate the MBP21.5 gene from that encoding the adult, brain-derived form of human MBP (18.5 kDa isoform; hMBP18.5). MBPs were expressed at high levels in E. coli and extracted from whole cells by simultaneous acid solubilization and mechanical disruption. A nearly two-fold increase in recombinant protein was detected in strains harboring MBP genes with bacterial preferred codons compared to genes containing human codons. The recombinant molecules were purified in two steps, first by reversed-phase chromatographic separation and then by metal affinity chromatography. Dimeric forms of recombinant MBP21.5 were detected under physiological conditions, however, substitution of a serine for the single cysteine at amino acid residue 81 resulted in only monomer formation. All forms of recombinant MBPs induced proliferative responses of human T lymphocytes specific for epitopes in MBP18.5 kDa. In contrast, human T cell lines that recognize an exon 2-encoded epitope of MBP responded to the 21.5 kDa isoform of MBP, but not the 18.5 kDa isoform.
我们已在细菌中设计并表达了人髓鞘碱性蛋白的重组胎儿形式（21.5 kDa异构体；rhMBP21.5），它是多发性硬化症中的一种候选自身抗原。外显子2插入、羧基末端组氨酸标签和优选的细菌密码子使MBP21.5基因与编码成人脑源性人MBP形式（18.5 kDa异构体；hMBP18.5）的基因有所不同。MBP在大肠杆菌中高水平表达，并通过同时酸溶解和机械破碎从全细胞中提取。与含有人密码子的基因相比，在携带具有细菌优选密码子的MBP基因的菌株中检测到重组蛋白增加了近两倍。重组分子通过两步纯化，首先通过反相色谱分离，然后通过金属亲和色谱。在生理条件下检测到重组MBP21.5的二聚体形式，然而，将氨基酸残基81处的单个半胱氨酸替换为丝氨酸仅导致单体形成。所有形式的重组MBP均诱导了针对MBP18.5 kDa表位的人T淋巴细胞增殖反应。相比之下，识别MBP外显子2编码表位的人T细胞系对MBP的21.5 kDa异构体有反应，但对18.5 kDa异构体无反应。
