Optimization and High Level Production of Recombinant Synthetic Streptokinase in E. coli Using Response Surface Methodology.

Streptokinase (SK) is an extracellular protein comprising 414 amino acids with considerable clinical importance as a commonly used thrombolytic agent. Due to its wide spread application and clinical importance designing more efficient SK production platforms worth investigation. In this regard, a synthetic SK gene was optimized and cloned in to pET21b plasmid for periplasmic expression. Response surface methodology was used to design a total of 20 experiments for optimization of IPTG concentration, post-induction period, and cell density of induction (OD600). The optimum levels of the selected parameters were successfully determined to be 0.28 mM for IPTG concentration, 9.889 H for post induction period, and 3.40768 for cell density (OD600). These settings result in 4.14fold increase in SK production rate of optimum expression conditions (7663 IU/mL) in comparison to the primary expression conditions (1853 IU/mL). Achieving higher yields of SK production in shake flask could lead to more cost effective industrial production of this drug which is the ultimate aim of SK production studies.

Selective reduction in glutaminase activity of l­Asparaginase by asparagine 248 to serine mutation: A combined computational and experimental effort in blood cancer treatment.

Type II l­asparaginase (l­ASNase) is an FDA approved enzyme drug with extensive applications for treatment of certain blood cancers. However, the therapeutic efficiency of this enzyme is hampered by its undesirable glutaminase activity. Given the pivotal role of this enzyme against cancer, designing engineered mutants with diminished glutaminase activity would be of great therapeutic interest. To this end, N248S mutation was selected as the potential mutation with beneficial effects. Various in silico analyses including MD simulation, molecular docking and QMMM studies were performed to assess the effects of N248S mutation on the activity of the enzyme. Thereafter, this mutation along with N248A, N248V and N248T mutations as controls were exerted in l­ASNase gene. The results from in silico analyses and experimental efforts indicated that N248S mutation is associated with the suitable l­ASNase activity, while the glutaminase activity is disturbed due to impaired interactions. It has been shown that glutamine turnover was affected much more strongly than asparagine hydrolysis. The approach of exploiting in silico tools to design mutated enzymes lead to staggering time and cost reduction. Following this strategy, we have designed a mutant l­ASNase with diminished glutaminase activity, which could be of interest for improved biomedical applications.