Expression And Purification In E.coli And Activity In Vitro Of Fusion Protein BDNF-TAT

Neurodegenerative diseases, such as Alzheimer's disease(AD), Parkinson's disease(PD), Amyotrophic lateral sclerosis(ALS) and Huntington's disease(HD), show movement disorders and learning or memory disorders in clinic, because of the death of neurons'in the brain.10 millions of people are suffered from AD and PD due to the absence of ideal therapy,which lead to heavy burden of Chinese people and society. A great deal of scientific research have proved that Neurotrophic Factors(NTFs) have a bright prospect in therapy of neurodegenerative diseases. Brain-derived neurotrophic factor(BDNF), one of NTFs, combines to its specific tyrosine kinase receptor trkB and then activates the downstream path and neurotrophy function.But blood-brain barrier(BBB) can prevent macromolecular drugs from passing into the brain. BBB is formed by a lot of compact vascular endothelial cells. There are blood cells inside of BBB and outside of BBB is adventitial cells, astrocytes, neurons. The reason why nature BDNF can not get into brain to play neurotrophy activities is the BBB.Protein transduction domain(PTD) is a structural domain containing rich positive charge, such as a kind of PTD from Trans-activator transcription(TAT) of HIV-1. Some compound, protein and nucleinic acid combined with PTD physically or chemically can pass through plasmalemma, nucleus even the BBB to play their activities. Some protein mediated by PTD can get through BBB and play its biologic activities, which can not pass into brain in normal.In the previous researches, we have succeeded in encoding maturate recombinant BDNF-TAT fusion protein gene in our experimental center. In order to cure neurodegenerative diseases, we hope to produce a new recombinant gene pharmaceutical preparation for the first time, which can pass through the BBB in the world.Based on the previous researches, the expression and purification of BDNF-TAT fusion protein in BL21(DE3)plysS E.coli were optimized in this study. The way of injection BDNF-TAT into caudal vein and promotion of neuron survival of BDNF-TAT fusion protein in vitro showed that BDNF-TAT can target into the brain and distribute in Hippocampal organization. This provided substantial material basis for further research about BDNF-TAT fusion protein in therapy of neurodegenerative diseases.Objectives:1.To optimize the protocol of expression and purification of BDNF-TAT fusion protein in the BL21(DE3)plysS E.coli.2.To study the distribution of BDNF-TAT fusion protein in the brain tissue after being injected into caudal vein.3.To study the neuron survival promoting effect of BDNF-TAT fusion protein in vitro.Methods:1.The recombinant vector PET-30(a)-BDNF-TAT was transfected into the BL21(DE3)plysS E.Coli,then the E.Coli was collected and suffered to ultrasonication. The E.Coli was inducted by the condition of 37℃, 1.0mM IPTG for 4 hours. All the samples were collected, then suffered to 15% SDS-PAGE and western blotting. Different induction temperatures (25℃,30℃,33℃,37℃,40℃), concentrations of IPTG (0.1 mM,0.5mM, 1.0mM,1.5mM), and induction time (1 hour,2 hours,4 hours,6 hours and 8hours) were tested for optimization. were also tested for optimization. The samples were collected and analyzed by 15% SDS-PAGE and Western Blotting to optimize the best induction condition of BDNF-TAT fusion protein. The recombinant vector PET-30(a)-BDNF-TAT was transformed into the BL21(DE3)plysS E.Coli and the the E.coli was colleted by high-speed centrifugation after being induced by the condition of 25℃with 0.5mM IPTG for 4 hours. Dispersed and washed by PBS, the E.coli was split in PBS by ultrasonication with the intensity of 60%. The supernatant was removed after centrifugating the splited E.coli by the condition of 12000rpm at 4℃. Precipitation was washed by 2M urea and 0.4% DOC by twice in turns. The supernatant was removed by low-temperature and high-speed centrifugation and the inclusion bodies was resolved by 8M urea with agitation for 14 hours at 4℃. The dissolved protein was isolated by SP-Sepharose cation exchange resin after pumped by mesohigh chromatographic system. To optimize the ionic strength of eluting of BDNF-TAT fusion protein,0.1 M and 0.5 M NaCl with pH 7.0 was used to wash the resin by the method of gradient elution. Finally, the washing way of 0.5 M NaCl with pH 8.5 occurred to isolate BDNF-TAT fusion protein. And then the interest protein was renatured by coubling dilution of 0.4 M Arginine to remove the urea. At last, we collected the samples above and analyzed the best purification conditions of BDNF-TAT fusion protein by 15% SDS-PAGE and Western Blot.2.Nine Kunming rats were divided into 3 groups randomly:drug group, negative control group and blank cantrol group(n=3). The renatured fusion protein BDNF-TAT which was dispersed in proper saline was injected into rats by 4μg in drug group by caudal vein, and the rats of saline control group were injected with equal-volume saline while the rats of blank control group being treated with nothing. To identify the BDNF, the heads of rats were Cut off to fetch the brain tissues after treated with drug for 4 hours and the whole protein was extracted from the brain tissues by holoprotein extraction kit.β-actin was used to be the Inner-Reference.3.Six Kunming rats was divided into 2 groups randomly:drug group and negative cantrol group(n=3). The renatured BDNF-TAT fusion protein were dispersed into proper saline and 4μg BDNF-TAT was injected into rats of drug group and equal-volume saline was injected into the rats of saline control group by caudal vein.All the heads of rats were cut off to fetch the brain tissues after treated by BDNF-TAT fusion protein for 4 hours. The tissues were soaked into 4% paraformaldehyde for 2 hours before being dipped into 30% sucrose for one night. The brain tissues freezed by -20℃were cut into the depth of lOum by freezing microtome. The distribution of BDNF-TAT in brain tissue was confirmed by the way of SABC immunohistochemisty.4.The dorsal root ganglions of One-week old SD rats were separated and digested into single neuron cells by collagenase IV and 0.25% trypsin. The neuron cells were divided into 3 groups:drug group, positive control group and blank cantrol group. Drug group was treated by DMEM culture media mixed with 100ng/ml BDNF-TAT, while positive control group was treated by DMEM culture media mixed with 100ng/ml NGF and blank cantrol group was treated by no neurotrophic factors in DMEM culture media. Exchanged the media every two days and the neuron cells was stained by AChE methods. Observed the growth of the neuron cells.Results: 1. A great quantity of BDNF-TAT fusion protein expressed in E.coli as inclusion bodies after transforming the recombinant vector PET-30(a)-BDNF-TAT into BL21(DE3)plysS with the Molecular Weight about 18kD by 15% SDS-PAGE and Western blotting. A lot of interest protein was found in precipitation after splited by ultrasonication and the results showed that BDNF-TAT fusion protein existed in E.coli as inclusion bodies. The condition of 25℃with 0.5mM IPTG for 4 hours was the best expression condition after optimization the three factors (induction temperature, concentration of IPTG, induction time).2. After being analyzed by 15% SDS-PAGE and Western blotting,most impurities in inclusion bodies were washed clear by 2M urea and 0.4% DOC. And the inclusion bodies were resolved in 8 M urea before being purified by a SP-Sepharose cation exchange resin. Most BDNF-TAT fusion protein exsisted in the elution of 0.5M NaCl with pH 8.5. And about 4.8mg of BDNF-TAT fusion protein in one litre E.coli was collected with a purity of 90% after renatured by 0.4M Arginine.3. The way of extracting the whole proteins from the brain tissue was conducted after tail vein injection of 4μg BDNF-TAT in Kunming mice for 3 hours.BDNF-TAT fusion protein targeted into the brain by the analysis of Western Bloting. The gray ratio between the gray ratio of BDNF-TAT and the gray ratio ofβ-actin stood the relative content of BDNF-TAT. The result (x±s) was:drug group 1.897±0.286, negative control group 0.615±0.234, blank control group 0.335±0.154. There is homogeneity of variance by Levene (P=0.447>0.05). There is significant difference of the longest lenth of axons between groups by One-way ANOVA (F=39.019, P=0.000). Multiple Comparisons by LSD:The relative content of BDNF-TAT in drug group was obvious higher than that in negative control group and blank control group (P<0.05) and there is no difference between negative control group and blank control group (P>0.05). It proved that BDNF-TAT can targeted into the brain of rat.4. There were obvious dying areas in the brain of rats in drug group confirmed by SABC immunohistochemisty of BDNF-TAT fusion protein, which distributed in CA1,CA3 and DG of hippocampus. And there was no obvious dying areas in negtive control group.Confirmed by AChE dying method, the results showed that the neuron cells grew better in drug group and positive group than that in blank group. Randomly chose 10 visual fields in each group to compare the longest lenth of axons of each group by Image Pro Plus 6.0. The result (x±s) was:drug groupl43±13μm,positive control group154±13μm,blank control group 64±15μm. There was homogeneity of variance by Levene (F=0.039,P=0.0.961>0.05). There was significant difference of the longest lenth of axons between groups by One-way ANOVA (F=126.523,P=0.000). Multiple Comparisions by LSD:There was no significant difference between drug group and positive control group (P=0.087>0.05).There was significant difference between drug group and blank control group (P=0.000<0.05). There was significant difference between positive control group and blank control group (P=0.000<0.05). Compared the total neuron cells area of each group by Image Pro Plus 6.0. The result (x±s) is:drug group 2686±242μm2, positive control group 2864±169μm2, blank control group 397±97μm2. There was no homogeneity of variance analysed by Levene (F=0.039, P=0.047<0.05). There was significant difference of the total neuron cells area between groups by analysed Welch (F=997.983, P=0.000). Multiple Comparisions by Dunnett T3:There was no significant difference between drug group and positive control group (P=0.199>0.05). There was significant difference between drug group and blank control group (P=0.000<0.05). There was significant difference between positive control group and blank control group (P=0.000<0.05). The results proved that BDNF-TAT fusion protein protected the neuron cells alive in vitro.Conclusion:1.The results implied that BDNF-TAT fusion protein could be expressed in E.coli as inclusion bodies after transforming the recombinant vector PET-30(a)-BDNF-TAT into BL21(DE3)plysS with the Molecular Weight about 18kD.2.Inclusion bodies were purified by cation exchange resins and BDNF-TAT were renatured by 0.4M Arginine. About 4.8mg BDNF-TAT fusion protein was produced with the purity of 90% in one litre E.coli.3.Western Bloting was used to analyze the relative contant of BDNF-TAT in the brain tissue of Kunming rats.The results showed that the relative content of BDNF-TAT in drug group was obvious higher than that in negative control group and blank control group(P<0.05). And there is no difference between negative control group and blank control group(P>0.05).It proved that BDNF-TAT targeted into the brain of rats.4.The results showed that BDNF-TAT fusion protein distributes in the CA1,CA3 and DG of hippocampus that confirmed by SABC immunohistochemisty.5.The neuron cells were cultivated with BDNF-TAT well.