Pillay Priyen, Kunert Karl J, van Wyk Stefan, Makgopa Matome Eugene, Cullis Christopher A, Vorster Barend J
a Department of Plant and Soil Sciences , Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria , Hillcrest, Pretoria , South Africa.
b Department of Biology , Case Western Reserve University , Cleveland , OH , USA.
Bioengineered. 2016 Nov;7(6):459-477. doi: 10.1080/21655979.2016.1208868. Epub 2016 Jul 26.
There is a growing interest in applying tobacco agroinfiltration for recombinant protein production in a plant based system. However, in such a system, the action of proteases might compromise recombinant protein production. Protease sensitivity of model recombinant foot-and-mouth disease (FMD) virus P1-polyprotein (P1) and VP1 (viral capsid protein 1) as well as E. coli glutathione reductase (GOR) were investigated. Recombinant VP1 was more severely degraded when treated with the serine protease trypsin than when treated with the cysteine protease papain. Cathepsin L- and B-like as well as legumain proteolytic activities were elevated in agroinfiltrated tobacco tissues and recombinant VP1 was degraded when incubated with such a protease-containing tobacco extract. In silico analysis revealed potential protease cleavage sites within the P1, VP1 and GOR sequences. The interaction modeling of the single VP1 protein with the proteases papain and trypsin showed greater proximity to proteolytic active sites compared to modeling with the entire P1-polyprotein fusion complex. Several plant transcripts with differential expression were detected 24 hr post-agroinfiltration when the RNA-seq technology was applied to identify changed protease transcripts using the recently available tobacco draft genome. Three candidate genes were identified coding for proteases which included the Responsive-to-Desiccation-21 (RD21) gene and genes for coding vacuolar processing enzymes 1a (NbVPE1a) and 1b (NbVPE1b). The data demonstrates that the tested recombinant proteins are sensitive to protease action and agroinfiltration induces the expression of potential proteases that can compromise recombinant protein production.
在基于植物的系统中，应用烟草农杆菌浸润法生产重组蛋白越来越受到关注。然而，在这样的系统中，蛋白酶的作用可能会影响重组蛋白的生产。研究了模型重组口蹄疫（FMD）病毒P1多聚蛋白（P1）和VP1（病毒衣壳蛋白1）以及大肠杆菌谷胱甘肽还原酶（GOR）对蛋白酶的敏感性。与用半胱氨酸蛋白酶木瓜蛋白酶处理相比，用丝氨酸蛋白酶胰蛋白酶处理时，重组VP1的降解更为严重。在农杆菌浸润的烟草组织中，组织蛋白酶L样、B样以及天冬酰胺内肽酶的蛋白水解活性升高，并且当与这种含蛋白酶的烟草提取物一起孵育时，重组VP1会被降解。计算机分析揭示了P1、VP1和GOR序列内潜在的蛋白酶切割位点。与用整个P1多聚蛋白融合复合物建模相比，单个VP1蛋白与蛋白酶木瓜蛋白酶和胰蛋白酶的相互作用建模显示其与蛋白水解活性位点的距离更近。当应用RNA测序技术利用最近可得的烟草草图基因组鉴定变化的蛋白酶转录本时，在农杆菌浸润后24小时检测到了几种差异表达的植物转录本。鉴定出了三个编码蛋白酶的候选基因，其中包括脱水响应蛋白21（RD21）基因以及编码液泡加工酶1a（NbVPE1a）和1b（NbVPE1b）的基因。数据表明，所测试的重组蛋白对蛋白酶作用敏感，并且农杆菌浸润会诱导可能影响重组蛋白生产的蛋白酶的表达。
