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首页> 外文期刊>Clinical and diagnostic laboratory immunology >Expression of recombinant feline tumor necrosis factor is toxic to Escherichia coli.
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Expression of recombinant feline tumor necrosis factor is toxic to Escherichia coli.

机译：重组猫肿瘤坏死因子的表达对大肠杆菌有毒。

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The tumor necrosis factor (TNF) genes from cats, horses, and pigs have all been cloned into the pFLAG-1 fusion protein expression vector (International Biotechnologies, Inc., Kodak, New Haven, Conn.). Growth curves for Escherichia coli containing the pFLAG-1 vector alone and the pFLAG-1 vector containing the TNF gene from each species were determined by visible light spectrophotometry (at 600 nm). Porcine TNF, equine TNF, and feline TNF cultures had slower doubling rates than cultures containing the pFLAG-1 vector alone. Cultures of cells transformed with feline TNF reached peak densities at 3 to 4 h and then decreased to near initial densities prior to the recovery of growth. The induction of expression with isopropyl-beta-D-thiogalactopyranoside (IPTG) arrested the growth of fresh feline TNF cultures for 6 h, which was followed by complete recovery. This inhibition occurred in two strains of E. coli (LL308 and JM101). Induced feline TNF cultures expressed the TNF-FLAG fusion protein for the first 6.5 h. Uninduced cultures expressed low levels of fusion protein. The feline TNF-pFLAG-1 vector was purified from cells expressing fusion protein and from cells with recovered growth curves. Sequencing the vector demonstrated the complete feline TNF gene and tac promoter in cells expressing the fusion protein and a deletional mutation of the tac promoter site in recovered cells. In contrast to equine and porcine TNF, the expression of recombinant feline TNF is toxic to E. coli. Alterations in protein folding and the prevention of secretion of the feline protein may explain the toxic effect.
机译：来自猫，马和猪的肿瘤坏死因子（TNF）基因已全部克隆到pFLAG-1融合蛋白表达载体（International Biotechnologies，Inc.，柯达，纽黑文，康涅狄格州）中。通过可见光分光光度法（在600nm）测定仅包含pFLAG-1载体的大肠杆菌和包含来自每种物种的TNF基因的pFLAG-1载体的生长曲线。猪TNF，马TNF和猫TNF培养物的加倍速率比仅包含pFLAG-1载体的培养物慢。用猫TNF转化的细胞培养物在3至4 h达到峰值密度，然后在恢复生长之前降低至接近初始密度。异丙基-β-D-硫代半乳糖吡喃糖苷（IPTG）诱导的表达使新鲜猫TNF培养物的生长停滞了6小时，然后完全恢复。这种抑制作用发生在两种大肠杆菌（LL308和JM101）中。诱导的猫TNF培养物在开始的6.5小时内表达TNF-FLAG融合蛋白。未诱导的培养物表达低水平的融合蛋白。从表达融合蛋白的细胞和具有恢复的生长曲线的细胞中纯化出猫TNF-pFLAG-1载体。对该载体进行测序证明了表达融合蛋白的细胞中完整的猫TNF基因和tac启动子，以及回收细胞中tac启动子位点的缺失突变。与马和猪TNF相反，重组猫TNF的表达对大肠杆菌有毒。蛋白质折叠的改变和猫蛋白质的分泌的阻止可能解释了毒性作用。

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