Correlation Between Thrombolytic Activity Of Plasmin QK And Amino Acid Sites And The Preliminary Study On Protein Crystal Structure

In recent years,with the continuous improvement of people's living standards,the prevalence of cardiovascular and cerebrovascular diseases has continued to increase.It is reported that 12 million people worldwide die of cardiovascular and cerebrovascular diseases every year.Countless people suffer from inconvenience caused by these diseases.The biggest cause of cardiovascular and cerebrovascular diseases is vascular embolism.The key to treatment is to clear the blood vessels to keep the blood flowing.At present,the clinical treatment drugs are mainly emergency drugs.There are shortcomings such as short-term effects and bleeding side effects.There is a tendency to develop new thrombolytic drugs.The research object is a new type of thrombolytic drug with great potential-Bacillus subtilis plasmin QK.It is a serine protease highly homologous to Nattokinase,which can target the degradation of the thrombus skeleton,cross-linked fibrin,with high thrombolytic activity and long drug half-life.Plasmin QK has a good application prospect as a new thrombolytic drug for the treatment of cardiovascular and cerebrovascular diseases.In the previous research of Professor Wang Yefu's group,a method to improve the fibrinolytic activity of QK was obtained by selecting wild-type strains and optimizing fermentation conditions.Purification was performed by gel filtration chromatography and other methods.Finally the purity was higher than 95%.The production process of plasmin QK is relatively mature,but the molecular mechanism of thrombolysis is still unclear.In this context,we explored the correlation between the amino acid sites of QK and enzyme activity.Preliminary analysis of protein crystal structure was conducted.The relationship between protein structure and thrombolytic function was explored.It provides a theoretical basis for the drug development of plasmin QK and a new drug choice for the prevention and treatment of cardiovascular and cerebrovascular diseases.The research contents are summarized into the following four aspects:?1?Fifteen strains are selected from B.subtilis fibrinolytic powder from different sources.Using the total DNA of these 15 strains as a template,their QK genes are amplified and sequenced.After that,they are aligned with the protein sequence corresponding to the QK gene submitted on Gen Bank?Gene Bank number:CAE18180.2?to find amino acid differences.According to the different mutation sites,15 strains are divided into four groups:A,B,C,and D.Among them,the mutation site of group A is S184T;the mutation sites of group B are Q90K,N193S,S236T,N365S.The mutation sites of group C are N193S,S236T,and A289V;the mutation sites of group D are Q90K,N193S,S236T,and A289V.?2?The fibrin plate method is used to determine the thrombolytic activity produced by the strain.The BCA protein assay kit is used to measure the total protein content of the fermentation broth.The ratio of the former to the latter is calculated to obtain the unit enzyme activity.The unit enzyme activity in group A is the highest?p<0.05?,with an average of 9.9 IU/mg;the average unit enzyme activities between groups B and C are 3.39 IU/mg and 2.69 IU/mg,respectively.There is no significant difference between the two groups?p>0.05?.But the unit enzyme activity of the two groups is significantly smaller than that of group A?p<0.05?.The average unit enzyme activity of group D is 0.19IU/mg,which is significantly lower than the other three groups?p<0.05?.?3?The strain with the highest unit enzyme activity is selected,and the protein produced is purified to obtain a high-purity plasmin QK that can be used for crystal culture.Crystal screening kit is used to select and optimize the crystal conditions using the hanging drop method.For protein crystals,the crystal culture temperature is16?.The protein concentration is 15mg/m L.And the bath conditions are 0.1M Bicine p H9.0,2%1,4-Dioxane,10%w/v polyethylene glycol?PEG?20,000,and 0.2M sodium fluoride?Na F?p H7.3,20%w/v PEG 3,350.?4?The obtained crystals are subjected to X-ray diffraction to obtain diffraction data.The resolution is 1.9?.Using HKL2000,CCP4,COOT,Phenix and other software for data processing and analysis,the crystal structure of plasmin QK is obtained.The plasmin QK crystal belongs to the P1 2₁1 space group.The unit cell belongs to the monoclinic crystal system.The unit cell parameter bond length is a=74.1?,b=79.4?,c=87.4?.The bond angle is?=90.0°,?=111.5°,?=90.0°.The integrity reaches 99.6%.This study explored the correlation between the thrombolytic activity of B.subtilis plasmin QK and amino acid sites.We identified amino acid sites that may have an effect on unit enzyme activity.The plasmin produced by B.subtilis with the highest enzyme activity was purified and crystallized to obtain high-quality protein crystals.The crystal structure was successfully resolved,which provided a theoretical basis for studying the thrombolytic activity mechanism of plasmin QK.