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首页> 外文期刊>Clinical and diagnostic laboratory immunology >Secretion of the mycobacterial 19-kilodalton protein by Escherichia coli, a novel method for the purification of recombinant mycobacterial antigens.
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Secretion of the mycobacterial 19-kilodalton protein by Escherichia coli, a novel method for the purification of recombinant mycobacterial antigens.

机译：大肠埃希菌分泌分枝杆菌19-千洛酮蛋白，这是一种纯化重组分枝杆菌抗原的新方法。

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We have reported previously (R.G. Hewinson and W.P. Russell, J. Gen. Microbiol. 139:1253-1259, 1993) the secretion by Escherichia coli of a recombinant form of the immunogenic protein MPB70 of Mycobacterium bovis when the protein is translated from its native initiation codon. N-terminal sequence analysis of the purified protein revealed that the signal peptide of MPB70 was cleaved by an endopeptidase of E. coli at the same cleavage site as reported for the protein in M. bovis. Since both the B- and T-cell antigenicities of the purified recombinant protein were similar to that of the native protein, the 19-kDa antigen of M. bovis was used as a model to test whether the signal peptide of MPB70 could direct the secretion of heterologous proteins in E. coli and whether antigen produced in this way retained antigenicity superior to that of recombinant protein produced as a fusion to glutathione-S-transferase. A chimeric protein was produced in which the signal peptide of MPB70 was fused to the 19-kDa antigen of M. bovis at amino acid residue 23. This chimeric protein was found to be secreted into the periplasm and culture medium of recombinant E. coli, and the signal peptide was cleaved by an endopeptidase of E. coli during secretion. Secretion of the 19-kDa antigen facilitated purification of the antigen by two-stage preparative electrophoresis which gave yields of 2.5 mg of purified, soluble 19-kDa antigen from 2.5 g (wet weight) of E. coli. Antigen purified in this way retained both B- and T-cell antigenicities. Moreover, the nonspecific mitogenic activity of the purified 19-kDa antigen was low, while the magnitude of the T-cell response induced by the purified antigen was considerably higher than that observed with purified antigen produced as a fusion protein with glutathione-S-transferase.
机译：以前我们已经报道过（RG Hewinson和WP Russell，J.Gen.Microbiol.139：1253-1259，1993），当从大肠杆菌中分离出牛分枝杆菌的免疫原性蛋白MPB70的重组形式时，大肠杆菌会分泌该蛋白。起始密码子。纯化蛋白的N-末端序列分析表明，MPB70的信号肽在与牛分枝杆菌中报道的蛋白相同的切割位点被大肠杆菌的内肽酶切割。由于纯化的重组蛋白的B细胞和T细胞抗原性均与天然蛋白相似，因此使用牛分枝杆菌的19 kDa抗原作为模型来测试MPB70的信号肽是否可以指导分泌大肠杆菌中的异源蛋白的鉴定以及以这种方式产生的抗原是否保留了比通过与谷胱甘肽-S-转移酶融合产生的重组蛋白更高的抗原性。产生了一种嵌合蛋白，其中MPB70的信号肽在氨基酸残基23处与牛分枝杆菌的19 kDa抗原融合。该嵌合蛋白被发现分泌到重组大肠杆菌的周质和培养基中，在分泌过程中，信号肽被大肠杆菌的内肽酶切割。 19-kDa抗原的分泌通过两步制备性电泳促进了抗原的纯化，这从2.5g（湿重）的大肠杆菌产生了2.5mg纯化的，可溶的19-kDa抗原。以这种方式纯化的抗原保留了B细胞和T细胞的抗原性。此外，纯化的19-kDa抗原的非特异性促有丝分裂活性较低，而纯化的抗原诱导的T细胞反应的幅度则明显高于用与谷胱甘肽S-转移酶融合蛋白生产的纯化抗原所观察到的水平。 。

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