| Luciferase has a wide range of applications in scientific research,among which it is the most studied as a reporter gene,most of which occurs in cells,and can also be applied to ATP detection,pyrosequencing,etc.Many mutant firefly luciferases have been developed to produce different bioluminescent colors,enzyme stability,or catalytic efficiency,however,these have not achieved significant improvements in luminous intensity and thermal stability,and still need to be improved.In this paper,the molecular modification of firefly luciferase in North America was mainly carried out by rational design,in order to obtain mutants with significantly improved thermal stability under the premise that the enzyme activity was not reduced compared with the original enzyme activity,and apply them to drug screening.Based on the rational design method of consensus sequence search and surface amino acid replacement,35 single point mutants were constructed and 6 single point mutants with improved thermostability and enzyme activity compared with wild type were found.They were T214 N,S201T,L530 I,A532R,T290 P,and K28 R,and the thermal stability of the corresponding mutants after 10 minutes in a 35°C metal bath was increased by 20.31,0.72,1.82,1.16,4.75,and 0.83 times,respectively.And the enzyme activities were respectively increased by 4.5,1.3,1.3,1.0,0.7,and 0.3 times compared with the wild type at room temperature.Iterative combinatorial mutations were carried out on them to obtain 27 combinatorial mutant luciferases,among which the residual activity of L530I/T214N/T290 P,S201T/L530I/T214N/T290 P,A532R/T214N/T290 P,T214N/T290 P after different incubating times(5-25 min)of metal bath at 35°C was determined,and the wild type was basically inactivated after 5 min of metal bath,while the residual activity of the corresponding mutants was still 82%,80%,57%,47%,respectively.Then the obtained optimal mutant L530I/T214N/T290 P was applied to drug screening cell line construction.The results showed that the L530I/T214N/T290 P mutant had higher sensitivity in drug screening than wild enzyme,and the IC50 value of the drug IL-6 was decreased from 1.309 to 1.034,which implied that the activity of the optimized luciferase mutant were improved. |
