expression and activity evaluation of reteplase in escherichia coli top10
نویسندگان
چکیده
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 l lb medium and induced by 0.02% l-arabinoseat 37°c, ph 7, and 180 rpm until od 600 of 0.6 was reached.samples were analyzed by sds-page and western blotting andthe expression of reteplase was examined. finally the activity of this recombinant protein was evaluated using chromogenic activity assay kit. the presence of reteplase in transformed escherichia coli top10 wasexamined by western blotting which revealed that the target protein in form inclusion body was expressed as a unique band at39 and the refolded reteplase was 66kda. the amount of protein produced was 90.5µg/ml and its activity was determined as 0.8 units. in this study, the expression of reteplase in escherichia coli top10 wasscaled up under optimum condition. furthermore we earned reteplase with partially suitable thrombolytic activity.
منابع مشابه
Cloning 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 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 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 کاملoptimization of the expression of reteplase in escherichia coli top10 using arabinose promoter
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 ...
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:
journal of paramedical sciencesجلد ۶، شماره ۳، صفحات ۰-۰
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