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首页> 外文期刊>Journal of Molecular Biology >Activity of foreign proteins targeted within the bacteriophage T4 head and prohead: Implications for packaged DNA structure
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Activity of foreign proteins targeted within the bacteriophage T4 head and prohead: Implications for packaged DNA structure

机译：靶向噬菌体T4头部和前部区域的外源蛋白质的活性：对包装的DNA结构的影响

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The phage-derived expression, packaging, and processing (PEPP) system was used to target foreign proteins into the bacteriophage capsid to probe the intracapsid environment and the structure of packaged DNA. Small proteins with minimal requirements for activity were selected, staphylococcal nuclease (SN) and green fluorescent protein (GFP). These proteins were targeted into the T4 head by means of IPIII (internal protein III) fusions or CTS (capsid targeting sequence) fusions. Additional evidence is provided that foreign proteins are targeted into T4 by the N-terminal ten amino acid residue consensus CTS of IPIII identified in previous work. Fusion proteins were produced within host bacteria by expression from plasmids or by production from recombinant phage carrying the fusion genes. Packaged fusion proteins CTS del IPIII del SN, CTS del IPIIITSN, CTS del IPIII del GFP, CTS del IPIII(T)GFP, and CTS del GFP, where del indicates a linkage peptide sequence Leu(Ile)-N-Glu cleaved by the T4 head morphogenetic proteinase gp21 during head maturation, are observed to exhibit intracapsid activity. SN activity within the head is demonstrated by loss of phage viability and by digested genomic DNA patterns visualized by gel electrophoresis when viable phage are incubated in Ca2+ Green fluorescent phage result immediately after packaging GFP produced at 30 degrees C and below, and continue to give green fluorescence under 470 nm light after CsCl purification. Non-fluorescent GFP-fusions are produced in bacteria at 37 degrees C, and phage packaged with these proteins achieve a fluorescent state after incubation for several months at 4 degrees C. GFP-packaged phage and proheads analyzed by fluorescence spectroscopy show that the mature head and the DNA-empty prohead package identical numbers of GFP-fusion proteins. Encapsidated GFP and SN can be injected into bacteria and rapidly exhibit intracellular activity. In vivo SN digestion of encapsidated DNA gives an intriguing pattern of DNA fragments by gel analysis, predominantly a repeat pattern of 160 bp multiples, reminiscent of a nucleosome digestion ladder, This quasi-limit DNA digestion pattern, reached >100-fold more slowly than the loss of titer, is invariant over a range less than or equal to 10 to 200 molecules of SN packaged per head, and independent of proteolytic cleavage of SN from the IPIII portion of the fusion, favoring a discontinuous packaged DNA structure. Rods of B-form DNA could be envisioned as protected from digestion, whereas bent or kinked DNA would be more susceptible to the diffusible SN. Such discontinuous packaged DNA structures are favored for phage T4 by a number of lines of evidence. (C) 1998 Academic Press. [References: 54]
机译：噬菌体衍生的表达，包装和加工（PEPP）系统用于将外源蛋白靶向噬菌体衣壳，以探查衣壳内环境和包装DNA的结构。选择对活性要求最低的小蛋白，葡萄球菌核酸酶（SN）和绿色荧光蛋白（GFP）。这些蛋白通过IPIII（内部蛋白III）融合或CTS（衣壳靶向序列）融合被靶向到T4头中。提供了其他证据，即先前工作中确定的IPIII的N末端十个氨基酸残基共有CTS将外源蛋白靶向到T4中。融合蛋白是通过质粒表达或携带融合基因的重组噬菌体在宿主细菌中产生的。包装的融合蛋白CTS del IPIII del SN，CTS del IPIIITSN，CTS del IPIII del GFP，CTS del IPIII（T）GFP和CTS del GFP，其中del表示被LPS切割的连接肽序列Leu（Ile）-N-Glu。观察到头部成熟过程中的T4头部形态发生蛋白酶gp21具有衣壳内活性。当将噬菌体在Ca2 +中孵育后，通过包裹噬菌体活力的丧失和凝胶电泳显示的消化的基因组DNA模式，可以证明头部内的SN活性。绿色荧光噬菌体在包装了30摄氏度及以下的GFP后立即产生绿色荧光噬菌体，并继续产生绿色CsCl纯化后在470 nm光下发出荧光。在37摄氏度的细菌中产生无荧光的GFP融合物，并且在4摄氏度下孵育几个月后，包装有这些蛋白质的噬菌体达到荧光状态。通过荧光光谱分析的GFP封装的噬菌体和前额表明成熟的头部和空DNA的前标包装相同数量的GFP融合蛋白。可以将衣壳化的GFP和SN注入细菌中，并迅速展现出细胞内活性。通过凝胶分析，对被包裹的DNA进行体内SN消化可得到有趣的DNA片段模式，主要是160 bp倍的重复模式，让人想起核小体消化阶梯。这种准极限DNA消化模式比100倍慢得多。滴度的损失在小于或等于每头包装的10到200个SN分子的范围内是不变的，并且独立于融合物IPIII部分中SN的蛋白水解切割，有利于不连续的包装DNA结构。可以预见，B形DNA的棒可防止消化，而弯曲或扭结的DNA对扩散性SN更敏感。许多证据表明，这种不连续的包装DNA结构被噬菌体T4所偏爱。 （C）1998年学术出版社。 [参考：54]

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