Afshari Elnaz, Amini-Bayat Zahra, Hosseinkhani Saman, Bakhtiari Nahid
Department of Microbiology, Islamic Azad University of Pharmaceutical Sciences Branch, Tehran, Iran.
Department of Biotechnology, Iranian Research Organization for Science and Technology (IROST), Tehran, Iran.
Avicenna J Med Biotechnol. 2017 Oct-Dec;9(4):169-175.
ATCC12633 can produce creatinase. It is a microbial enzyme which degrades creatinine in bacteria and provides source of carbon and nitrogen. Also, this enzyme is used in the enzymatic measurement of creatinine concentration for diagnosis of renal and muscles functions and diseases. Our purpose was recombinant production of creatinase for using in clinical measurement of serum or urine creatinine.
A 1209bp of open reading frame of creatinase was amplified by PCR from genome and cloned into pET28a expression vector which was digested using NheI and XhoI restriction enzymes. Cloning was confirmed by colony PCR, double digestion analysis and sequencing. Recombinant pET28a vector was transformed to . Creatinase expression was induced in using IPTG and confirmed by SDS-PAGE and western blotting. Purification of creatinase was performed using Ni-NTA column. The specific activity of this enzyme was also investigated.
The creatinase gene cloning was confirmed by DNA sequencing. Successful expression of creatinase was performed in (57.4% of total protein). SDS-PAGE and western blot analysis showed a 45 creatinase protein. Purification of creatinase was done with high purity. The specific activity of recombinant enzyme is 26.54 that is much higher than other creatinase used in the commercial kits (9 ).
The recombinant creatinase was expressed efficiently in and 57% of total protein was the recombinant creatinase. Also, expressed creatinase has high solubility and also the enzyme has good activity compared to enzymes used in commercial kits, so a new source of creatinase was produced for creatinine assay kit in this study.
ATCC12633可产生肌酸酶。它是一种微生物酶，能降解细菌中的肌酐并提供碳源和氮源。此外，该酶还用于酶法测定肌酐浓度，以诊断肾脏和肌肉功能及疾病。我们的目的是重组生产肌酸酶，用于血清或尿液肌酐的临床检测。
通过PCR从基因组中扩增出1209bp的肌酸酶开放阅读框，并克隆到用NheI和XhoI限制性内切酶消化的pET28a表达载体中。通过菌落PCR、双酶切分析和测序确认克隆。将重组pET28a载体转化至……。用IPTG在……中诱导肌酸酶表达，并通过SDS-PAGE和蛋白质印迹法进行确认。使用镍-氮三乙酸（Ni-NTA）柱对肌酸酶进行纯化。还对该酶的比活性进行了研究。
通过DNA测序确认了肌酸酶基因的克隆。在……中成功表达了肌酸酶（占总蛋白的57.4%）。SDS-PAGE和蛋白质印迹分析显示有一条45kDa的肌酸酶蛋白条带。肌酸酶得到了高纯度的纯化。重组酶的比活性为26.54U/mg，远高于商业试剂盒中使用的其他肌酸酶（9U/mg）。
重组肌酸酶在……中高效表达，且重组肌酸酶占总蛋白的57%。此外，表达的肌酸酶具有高溶解性，与商业试剂盒中使用的酶相比，该酶具有良好的活性，因此本研究为肌酐检测试剂盒生产了一种新的肌酸酶来源。
