Nguyen Sy Le Thanh, Quyen Dinh Thi, Vu Hong Diep
Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, District of Cau Giay, Hanoi 10600, Vietnam.
Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, District of Cau Giay, Hanoi 10600, Vietnam ; Department of Biotechnology and Pharmacology, University of Science and Technology of Hanoi, 18 Hoang Quoc Viet Road, District of Cau Giay, Hanoi 10600, Vietnam.
Biomed Res Int. 2014;2014:324705. doi: 10.1155/2014/324705. Epub 2014 May 5.
The streptokinase (SK) is emerging as an important thrombolytic therapy agent in the treatment of patients suffering from cardiovascular diseases. We reported highly effective renaturation of a SK from S. pyogeness DT7 overexpressed in E. coli, purification, and biochemical characterization. A gene coding for the SK was cloned from S. pyogeness DT7. Because accumulation of active SK is toxic to the host cells, we have expressed it in the form of inclusion bodies. The mature protein was overexpressed in E. coli BL21 DE3/pESK under the control of the strong promoter tac induced by IPTG with a level of 60% of the total cell proteins. The activity of the rSK, renatured in phosphate buffer supplemented with Triton X-100 and glycerol, was covered with up to 41 folds of its initial activity. The purified of protein was identified with MALDI-TOF mass spectrometry through four peptide fragments, which showed 100% identification to the corresponding peptides of the putative SK from GenBank. Due to overexpression and highly effective renaturation of large amounts of inclusion bodies, the recombinant E. coli BL21 DE3/pESK system could be potentially applied for large-scale production of SK used in the therapy of acute myocardial infarction.
链激酶（SK）正成为治疗心血管疾病患者的一种重要溶栓治疗药物。我们报道了从在大肠杆菌中过表达的化脓性链球菌DT7中高效复性SK、纯化及生化特性鉴定的过程。从化脓性链球菌DT7中克隆了编码SK的基因。由于活性SK的积累对宿主细胞有毒性，我们以包涵体的形式表达它。成熟蛋白在IPTG诱导的强启动子tac的控制下，于大肠杆菌BL21 DE3/pESK中过表达，表达量占总细胞蛋白的60%。在补充有Triton X-100和甘油的磷酸盐缓冲液中复性的重组链激酶（rSK）的活性比其初始活性提高了41倍。通过四个肽段用基质辅助激光解吸电离飞行时间质谱法（MALDI-TOF）鉴定纯化的蛋白，结果显示其与GenBank中推定的SK的相应肽段有100%的一致性。由于大量包涵体的过表达和高效复性，重组大肠杆菌BL21 DE3/pESK系统有可能用于大规模生产用于急性心肌梗死治疗的SK。
