Hafizi Akram, Malboobi Mohamad Ali, Jalali-Javaran Mokhtar, Maliga Pal, Alizadeh Houshang
Department of Plant Breeding and Biotechnology, Faculty of Agriculture, Tarbiat Modares University, P.O. Box 14115-336, Tehran, Iran.
Department of Plant Biology and Pathology, Rutgers, The State University of New Jersey, 59 Dudley Road, New Brunswick, NJ, 08901, USA.
Biotechnol Lett. 2017 Nov;39(11):1683-1688. doi: 10.1007/s10529-017-2416-0. Epub 2017 Aug 23.
To develop a deliberately engineered expression and purification system for an active chimeric-recombinant tissue plasminogen activator (crtPA) using co-expression with polyhydroxybutyrate (PHB) operon genes.
Fusion of crtPA with PhaC-synthase simplified the purification steps through crtPA sedimentation with PHB particles. Moreover, the covalently immobilized crtPA was biologically active as shown in a chromogenic assay. Upon WELQut-protease activity, the released single-chain crtPA converted to the two-chain form which produced a pattern of bands with approx. MW of 32 and 11 kDa in addition to the full length crtPA.
Fusion of crtPA with PhaC-synthase not only simplifies purification from the bacterial host lysate, but also co-expression of PHB operon genes creates an oxidative environment, thereby reducing the inclusion body formation possibility. The isolated crtPA-PHB granules exhibited crtPA serine protease activity. Thus, fusion with the PhaC protein could be used as a scaffold for covalent displaying of functional disulfide-rich proteins.
利用与聚羟基丁酸酯（PHB）操纵子基因共表达，开发一种用于活性嵌合重组组织纤溶酶原激活剂（crtPA）的精心设计的表达和纯化系统。
crtPA与PhaC合酶的融合通过crtPA与PHB颗粒的沉降简化了纯化步骤。此外，如显色测定所示，共价固定的crtPA具有生物活性。在WELQut蛋白酶活性作用下，释放的单链crtPA转化为双链形式，除全长crtPA外，还产生了约32 kDa和11 kDa的条带模式。
crtPA与PhaC合酶的融合不仅简化了从细菌宿主裂解物中的纯化，而且PHB操纵子基因的共表达创造了一个氧化环境，从而降低了包涵体形成的可能性。分离的crtPA-PHB颗粒表现出crtPA丝氨酸蛋白酶活性。因此，与PhaC蛋白的融合可作为共价展示富含功能性二硫键蛋白的支架。
