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首页> 外文期刊>BMC Molecular Biology >Multigene expression of protein complexes by iterative modification of genomic Bacmid DNA
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Multigene expression of protein complexes by iterative modification of genomic Bacmid DNA

机译：通过重复修饰基因组芽孢杆菌DNA蛋白质复合物的多基因表达

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Background Many cellular multi-protein complexes are naturally present in cells at low abundance. Baculovirus expression offers one approach to produce milligram quantities of correctly folded and processed eukaryotic protein complexes. However, current strategies suffer from the need to produce large transfer vectors, and the use of repeated promoter sequences in baculovirus, which itself produces proteins that promote homologous recombination. One possible solution to these problems is to construct baculovirus genomes that express each protein in a complex from a separate locus within the viral DNA. However current methods for selecting such recombinant genomes are too inefficient to routinely modify the virus in this way.Results This paper reports a method which combines the lambda red and bacteriophage P1 Cre-recombinase systems to efficiently generate baculoviruses in which protein complexes are expressed from multiple, single-locus insertions of foreign genes. This method is based on an 88 fold improvement in the selection of recombinant viruses generated by red recombination techniques through use of a bipartite selection cassette. Using this system, seven new genetic loci were identified in the AcMNPV genome suitable for the high level expression of recombinant proteins. These loci were used to allow the recovery two recombinant virus-like particles with potential biotechnological applications (influenza A virus HA/M1 particles and bluetongue virus VP2/VP3/VP5/VP7 particles) and the mammalian chaperone and cancer drug target CCT (16 subunits formed from 8 proteins).Conclusion 1. Use of bipartite selections can significantly improve selection of modified bacterial artificial chromosomes carrying baculovirus DNA. Furthermore this approach is sufficiently robust to allow routine modification of the virus genome. 2. In addition to the commonly used p10 and polyhedrin loci, the ctx, egt, 39k, orf51, gp37, iap2 and odv-e56 loci in AcMNPV are all suitable for the high level expression of heterologous genes. 3. Two protein, four protein and eight protein complexes including virus-like particles and cellular chaperone complexes can be produced using the new approach.
机译：背景技术许多细胞多蛋白复合物自然以低丰度存在于细胞中。杆状病毒表达提供了一种产生毫克量的正确折叠和加工的真核蛋白复合物的方法。然而，当前的策略遭受需要产生大的转移载体的需要，并且在杆状病毒中使用重复的启动子序列，杆状病毒本身产生促进同源重组的蛋白质。解决这些问题的一种可能的方法是构建杆状病毒基因组，该杆状病毒基因组从病毒DNA内的一个单独位点以复合物形式表达每种蛋白质。然而，目前选择此类重组基因组的方法效率太低，无法以这种方式进行常规修饰。结果本文报道了一种结合λ红色和噬菌体P1 Cre重组酶系统有效产生杆状病毒的方法，其中杆状病毒由多种复合蛋白表达，外源基因的单基因座插入。该方法基于通过使用二分选择盒对红色重组技术产生的重组病毒的选择提高了88倍。使用该系统，在AcMNPV基因组中确定了七个新的遗传基因座，适用于重组蛋白的高水平表达。这些基因座用于回收具有潜在生物技术应用的两个重组病毒样颗粒（甲型流感病毒HA / M1颗粒和蓝舌病毒VP2 / VP3 / VP5 / VP7颗粒）以及哺乳动物分子伴侣和癌症药物靶向CCT（16个亚基）由8种蛋白质组成。）结论1.使用两部分选择可以显着改善带有杆状病毒DNA的修饰细菌人工染色体的选择。此外，这种方法足够健壮，可以进行病毒基因组的常规修饰。 2.除常用的p10和多面体基因座外，AcMNPV中的ctx，egt，39k，orf51，gp37，iap2和odv-e56基因座均适用于高水平表达异源基因。 3.使用这种新方法可以生产出两种蛋白，四种蛋白和八种蛋白复合物，包括病毒样颗粒和细胞伴侣复合物。

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