optimization of the expression of reteplase in escherichia coli top10 using arabinose promoter
نویسندگان
چکیده
conclusions reteplase was expressed in e. coli top 10 after activation of pbad/giiia promoter region by arabinose and optimized. results the obtained recombinant plasmid was sequenced to confirm the presence and correct framing of reteplase gene regarding the expression of reteplase. maximum production of this enzyme was obtained under the following condition: 0.0002% l-arabinose at 37°c for 2 hours incubation. the purified protein was detected on sds-page (sodium dodecyl sulfate polyacrylamide gel electrophoresis) as a 66 kda band. the concentration of t-pa standard was 1 unit which is equal to 12 µg/ml. the enzymatic activity of samples was measured as 0.8 units compared to the standards. materials and methods the recombinant plasmid, pet15b/reteplase was digested by ncoi and bamhi restriction enzymes; while pbad/giiia vector was digested by ncoi and bglii. then the insert and vector were ligated and used for transformation of e. coli top10 cells by heat shock method. overnight culture of transformed bacteria was induced by l-arabinose in various concentrations (0.2, 0.02, 0.002, and 0.0002%) and at various temperatures. objectives this study aimed at expression of reteplase in optimum condition. in this study, the reteplase gene was cloned and expressed in escherichia coli top 10 as a suitable host cell and its expression was optimized. background reteplase is a mutant version of t-pa (tissue plasminogen activator) with prolonged half-life. in the present study, e. coli top 10 bacteria were utilized in the production of reteplase, which is the nonglycosylated active domain of t-pa. reteplase gene was ligated into pbad/giii plasmid which, allows secretion of this protein in periplasmic space. it would allow the correct formation of disulfide bonds in protein structure.
منابع مشابه
Optimization of the Expression of Reteplase in Escherichia coli TOP10 Using Arabinose Promoter
BACKGROUND Reteplase is a mutant version of t-PA (tissue plasminogen activator) with prolonged half-life. In the present study, E. coli Top 10 bacteria were utilized in the production of reteplase, which is the nonglycosylated active domain of t-PA. Reteplase gene was ligated into pBAD/gIII plasmid which, allows secretion of this protein in periplasmic space. It would allow the correct formatio...
متن کاملCloning and Expression of Functional Reteplase in Escherichia coli TOP10
BACKGROUND Production of tissue Plasminogen Activator protein (t-PA) in prokaryotes systems has many problems such as the lack of active protein production, multiple purification steps, and renaturation process which has been shown to be costly and time-consuming. METHODS In this study, reteplase which is the nonglycosylated active domain of t-PA was used to transform TOP10 Escherichia coli (...
متن کاملexpression and activity evaluation of reteplase in escherichia coli top10
reteplase is a part of tissue plasminogen activator (t-pa) used for theremoval of thrombi in blood vessels. in the present study we express the reteplase genein escherichia coli top10 and then its thrombolytic activity was measured. the recombinant plasmid pbadgiiia was transformed into the competent escherichia coli top10 and then transformed bacteria was seeded into bioreactor containing 1.5 ...
متن کاملcloning and expression of functional reteplase in escherichia coli top10
background: production of tissue plasminogen activator protein (t-pa) in prokaryotes systems has many problems such as the lack of active protein production, multiple purification steps, and renaturation process which has been shown to be costly and time-consuming. methods: in this study, reteplase which is the nonglycosylated active domain of t-pa was used to transform top10 escherichia coli (...
متن کاملoptimization of the expression of reteplase in escherichia coli
reteplase is a segment of tissue plasminogen activator used for the removal of thrombi in blood vessels. in the present study the cloned reteplase gene was used for its expression in competent e. coli. the recombinant plasmid, pet15b/reteplase (rpet-bl21), was transformed into competent e.coli strain bl21 (de3) cells. overnight culture of the transformed bacteria was induced by the addition of...
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عنوان ژورنال:
jundishapur journal of natural pharmaceutical productsجلد ۱۰، شماره ۱، صفحات ۰-۰
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