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首页> 外文期刊>Turkish Journal of Veterinary and Animal Sciences >Comparison of the efficiency of concentrated soluble recombinant phospholipase D and natural phospholipase D enzymes
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Comparison of the efficiency of concentrated soluble recombinant phospholipase D and natural phospholipase D enzymes

机译：浓缩的可溶性重组磷脂酶D和天然磷脂酶D酶的效率比较

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Phospholipase D (PLD) is a major virulence determinant of Corynebacterium pseudotuberculosis. Various studies have focused on the expression of recombinant PLD (rPLD) enzyme in different Escherichia coli strains, but generally a high yield of insoluble protein was reported. The aims of this study were to express soluble rPLD by different methods in E. coli. The rPLD and natural PLD (dPLD) enzymes were concentrated using an ultramembrane cassette system after the efficiencies of these concentrated enzymes were compared. The rPLD enzyme was expressed in One Shot?BL21(DE3) E. coli when induced by IPTG in TY medium. Soluble dPLD and rPLD enzyme hemolytic activities were determined using the reverse CAMP test. The nucleotide sequence of the rPLD gene was 99.7% similar to the PLD gene of C. pseudotuberculosis in the NCBI GenBank Database; these differences in nucleotides resulted in a difference in two amino acids. The rPLD protein concentration and the titer of hemolytic activity were 23.1 mg/mL and 1/256, respectively. Similarities in the enzyme characteristics were detected between rPLD and dPLD enzymes. These findings indicate that a protocol would be useful for the enhanced production of soluble rPLD in E. coli and a membrane cassette system for concentration the recombinant protein.
机译：磷脂酶D（PLD）是假结核棒杆菌的主要毒力决定因素。各种研究集中于重组PLD（rPLD）酶在不同大肠杆菌菌株中的表达，但通常报道了高产量的不溶蛋白。这项研究的目的是通过不同的方法在大肠杆菌中表达可溶性rPLD。比较了这些浓缩酶的效率后，使用超膜盒系统浓缩了rPLD和天然PLD（dPLD）酶。当IPTG在TY培养基中诱导时，rPLD酶在One Shot？BL21（DE3）大肠杆菌中表达。使用反向CAMP测试确定可溶性dPLD和rPLD酶的溶血活性。 rPLD基因的核苷酸序列与NCBI GenBank数据库中假结核假单胞菌的PLD基因相似，为99.7％。这些核苷酸差异导致两个氨基酸的差异。 rPLD蛋白浓度和溶血活性滴度分别为23.1 mg / mL和1/256。在rPLD和dPLD酶之间检测到酶特性的相似性。这些发现表明，对于在大肠杆菌和用于浓缩重组蛋白的膜盒系统中提高可溶性rPLD的产量的方案将是有用的。

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