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Expression and Purification in E.coli and Activity in Vitro of Fusion Protein BDNF-TAT

Author: LuoDaoFei
Tutor: JiAiMin
School: Southern Medical University,
Course: Pharmacy
Keywords: Brain-derived neurotrophic factor fusion protein Expression Purification Active
CLC: R741
Type: Master's thesis
Year: 2011
Downloads: 50
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Abstract

Neurodegenerative diseases, such as senile dementia (Alzheimer 's's disease, AD), Parkinson's disease (Parkinson,'s disease, PD), atrophic side cable sclerosis (Amyotrophic lateral sclerosis, ALS), and Huntington's disease (Huntington's disease HD), etc., clinical performance of limb movement and learning and memory dysfunction, mainly due to the absence or death of neuronal function. There is no ideal treatment for the two diseases in our AD and PD involving about 1,000 people to bring a heavy burden to individual families and society. The study shows that the members of the family of neurotrophic factors (neurotrophic factors, NTFs) and other well neurodegenerative diseases of the central nervous system therapeutic prospects. The brain-derived neurotrophic factor (BDNF) is a neurotrophic factor family members, including one of its specific receptor tyrosine kinase family members trkB combined activation of downstream signal transduction pathways, and play a neurotrophic biological activity. However, the blood-brain barrier (BBB) ??hinders macromolecular drugs from the blood into the brain play a physiological role in human brain blood-brain barrier (BBB) ??by the vascular endothelial cell tight junctions composed BBB cavity surface blood chamber outside for outer membrane cells, star line cells and a small number of neurons. Natural BDNF can not play through the BBB to enter the brain neurotrophic activity. Protein transduction domain (protein transduction domain, PTD), as derived from the the type Ⅰ human immunodeficiency virus transcriptional activator protein (Trans-activator transcription, the TAT)'s PTD, is rich in positive charge to its physical or chemical connection compounds, proteins, or nucleic acid molecules with the cell membrane into the cytoplasm, nucleus, or even BBB, play the structural domains of the respective biological function. PTD / TAT-mediated protein can cross the BBB by cerebrovascular endothelial cells into the brain play a physiological role of the carrier protein, leaving some in normal circumstances is not the protein through the BBB into the brain tissue. Early experimental center by genetic engineering methods successfully prepared fusion protein gene coding for the mature BDNF-TAT, aimed in the world is first prepared a through the BBB into the central nervous system, the treatment of neurodegenerative diseases recombinant neurotrophic factor pharmaceutical preparations. Based on preliminary studies, the use of recombinant plasmid PET-30 (a)-BDNF-TAT was transformed into E. coli BL21 (DE3) plysS optimization expression optimization, purification and arginine-fold dilution law renaturation BDNF-TAT cation exchange resin fusion protein KM mice after tail vein injection of brain targeting its distribution in the brain tissue, in vitro cultured The SD rats newborn one week rat dorsal root ganglion neurons detect BDNF-TAT fusion protein biology activity, provide the material basis for the further study of the treatment of neurodegenerative diseases through the blood-brain barrier drug BDNF-TAT fusion protein. The purpose. Research the recombinant plasmids PET-30 (a)-BDNF-TAT was transformed into E. coli BL21 (DE3) plysS BDNF-TAT fusion protein expression and optimization of BDNF-TAT fusion protein expressed in E. coli and purified ; 2. give renaturation mice by tail vein BDNF-TAT fusion protein, to explore in the brain tissue of mice brain targeting and distribution in the brain tissue of mice; 3. vitro cultured neonatal rat dorsal root ganglion neuron cells, further study refolded BDNF-TAT fusion protein in vitro to promote neuronal survival activity of the growth. Method 1. (A)-BDNF-TAT recombinant plasmid PET-30 prepared according to the the preliminary research group by the genetic engineering approach and, in this article the recombinant plasmid was transformed into E. coli BL21 (DE3) plysS 37 ° C, 1.0 mM IPTG was induced 4h The cells were harvested after low temperature (4 ℃, 5000 rpm) centrifugation, sonication, samples collected after each step in the high-speed centrifugal by 15% SDS-PAGE and Western Blot analysis of and determining fusion protein BDNF-TAT forms of expression in E. coli; respectively. Select 25 ° C, 30 ° C, 33 ° C, 37 ° C 40 ° C for different induction temperature, induction temperature optimized; were selected 0.1 mM, 0.5 mM, 1.0 mM, 1.5mM IPTG induction concentration of IPTG induction concentration by line optimization; selected 1,2,4,6,8 induction time, induction time optimized E. coli; collected under various conditions by the line 15% SDS-PAGE and (?) Western Blot analysis to determine BDNF-TAT fusion protein optimal expression conditions in E. coli. Two recombinant plasmids were transformed into E. coli, 25 ° C, induced by 0.5 mM IPTG after 4h was centrifuged to collect wet cells, PBS Resuspend washed with 60% strength sonication instrument cracking coli, 4 ° C, 12000rpm cryogenic high-speed high-speed centrifugation to the supernatant, the precipitate was washed with 2M urea and 0.4% the doc washed and repeatedly washed once, low-temperature high-speed centrifugation and the supernatant, inclusion bodies precipitated 8M urea 4 ° C dissolved with stirring 14H, dissolved proteins by high-pressure chromatography system pump into the SP- -Sepharose cation exchange column select pH7.0 NaCl concentrations of 0.1M and 0.5M eluant line gradient elution to optimize the ability of different ionic strength of the elution of the target protein; selected pH8.5 NaCI concentration of 0.5M eluent target protein, of hanging column by the line of the target protein separation and purification. The target protein eluted with 0.4M arginine-fold dilution method to remove the urea in the target protein, while refolding fusion protein BDNF-TAT. Collect various steps of protein samples 15% SDS-PAGE and Western Blot analysis to determine the final column purification optimization program. Randomly selected 9 KM mice as experimental subjects, divided into the administration group, negative control group and blank control group, n = 3, dissolved in an appropriate amount of saline the BDNF-TAT fusion protein purified refolded dose group animals tail vein injection 4μg BDNF-TAT, while the negative control group received an equal volume of saline, blank control group were not administered. 4h after decapitation downtown organization, full of protein extraction kit to extract the total protein in the brain tissue, immunoblotting (Western Blot) identification of brain tissue contained BDNF-TAT target protein in all groups on the p-actin as a loading control after the standardization of the sample volume, the ratio of the gray value to BDNF-TAT and β-actin the BDNF-TAT relative content in brain tissue, a one-way analysis of variance methods, compare administration group, negative control group HE between the blank group BDNF- The relative content of TAT whether there is statistically significant. Randomly selected 6 KM mice as experimental subjects, divided into treatment group and negative control group, n = 3, the purified refolded the BDNF-TAT dissolved in an appropriate amount of saline administered animals tail vein injection of BDNF-TAT, the negative control group received an equal volume of saline hours after perfusion of 4% paraformaldehyde, decapitated, the brain tissue, the brain tissue of mice 4% paraformaldehyde immersed in 30% sucrose solution after 2h soaking overnight, 10μm The thickness of the frozen section. Sliced ??to identify BDNF-TAT distribution in the brain tissue of mice with the SABC immunohistochemical method. 5. Removal of newborn SD rat dorsal root ganglion in a week or so after collagenase Ⅳ and 0.25% trypsin containing 10? S DMEM medium in vitro cultured dorsal root ganglion neurons. Neurons divided into dose group, positive control group and blank control group, DMEM medium dose group join 100ng/ml renaturation fusion protein BDNF-TAT 100ng/ml NGF the DMEM medium Add in the positive control group The DMEM medium not to join the blank control group neurotrophic factor. The medium was changed every two days once fluid and cultured for 4 days with AChE neurons dyeing method of neurons by line staining to observe the growth of neurons. 1 were identified by 15% SDS-PAGE and Western Blot BDNF-TAT, E. coli the BDNF-TAT fusion protein in the molecular weight of about 18kD at expression of the target protein in E. coli lysis precipitate more described BDNF-TAT fusion protein precipitation in the form of inclusion bodies exist; optimized by the different temperature, IPTG induction concentration and induction time through the line, induced by the temperature at 25 ° C, IPTG induction concentration 0.5mmM induced 4h the BDNF-TAT fusion protein expression in E. coli highest. By 15% SDS-PAGE and Western Blot analysis, inclusion body protein after 2M urea and 0.4% DOC wash most of the contaminating proteins are washed clean, and 8M urea dissolved most of inclusion body proteins dissolved protein SP- -Sepharose cation exchange column purification, the majority of the target protein is pH 8.5, NaCl concentration of 0.5 M eluate eluted a, 0.4M arginine refolded protein purity of about 90% per liter of bacteria Nengchan approximately 4.8 mg of the target protein. Western Blot Identification of brain tissue relative content of BDNF-TAT grayscale scanning the ECL light emitting developing film in the administration group, negative control group and blank control group BDNF-TAT and β-actin, BDNF-TAT and p gray-actin ratio as groups in BDNF-TAT protein relative expression levels of results as x ± s, the relative expression level of BDNF-TAT protein administered group was 1.897 ± 0.286, BDNF-TAT negative control group The relative expression level of the protein was 0.615 ± 0.234, the BDNF-TAT protein blank control group relative expression level was 0.335 ± 0.154. Levene's test of homogeneity of variance (P = 0.447 gt; 0.05), homogeneity of variance. Single direction variance analysis, each group between BDNF-TAT protein relative expression of the amount of the difference (F = 39.019, P = 0.000 lt; 0.05), the difference was statistically significant. Multiple comparisons: LSD method group administered group the BDNF-TAT relative expression of BDNF administration group in the amount of the negative control group, BDNF-TAT relative expression levels compared (P = 0.000 lt; 0.05), the difference was statistically significant; BDNF-TAT-TAT relative expression amount of blank control group compared to the relative expression level (P = 0.000 lt; 0.05), the difference was statistically significant; BDNF-TAT (?) head in the negative control group, the expression levels and gaps BDNF-TAT control group compared to the relative expression level (P = 0.188 gt; 0.05), the difference was not statistically significant, indicating that BDNF-TAT targeting to the brain tissue of mice by peripheral administration. 4.SABC immune histochemical method identification of BDNF-TAT fusion protein BDNF-TAT-positive staining in brain tissue distribution, administration group after tail vein injection KM mice significantly stronger positive saline group The staining was significantly weaker than the administration group, the staining was mainly distributed in the hippocampus CA1, CA3 and DG region. The AChE staining neurons, the cells of the control group neurons cultured 4 days after the smaller cell bodies, cell death processes shorter. Administration group and positive control group, the growth of neurons, the projection is longer, the number of cells is significantly higher than the blank control group. Each set of holes randomly selected 10 fields of view to measuring the longest neurite length of cells, Image Pro Plus6.0 software results to x ± s results dose group was 143 ± 13um, positive control group was 154 ± 13um, while blank control group was 64 ± 15um, Levene test of homogeneity of variance (F = 0.039, P = 0.961 gt; 0.05), the homogeneity of variance. Single direction variance analysis, longest neurite length of each group are different (F = 126.523, P = 0.000), and the difference was statistically significant. Multiple comparison LSD method groups: group administration longest neurite longest neurite length compared to the length of the positive control group (P = 0.087 gt; 0.05), the difference was not statistically significant, while the longest process administered group longest neurite length compared to the length of the blank control group (P = 0.000 lt; 0.05), the difference was statistically significant; longest process in the positive control group compared to the length of the longest neurite length and blank control group (P = 0.000 lt; 0.05), the difference was statistically significant, indicating that BDNF-TAT can promote neurite growth in vitro. Measuring the total area of ??the cell bodies of neurons by Image Pro Plus6.0 software the administration group 2686 ± 242um2, positive control group for 2864 ± 169um2, while the control group was 397 ± 97um2. Levene's test of homogeneity of variance (F = 0.039, P = 0.047 lt; 0.05), heterogeneity of variance. Welch analysis, the total area of ??the cell bodies of neurons in each group difference (F = 997.983, P = 0.000), the difference was statistically significant. Multiple comparisons: Dunnett T3 method group administered group compared to the total area of ??the cell bodies of neurons in the total area of ??the cell bodies of neurons and the positive control group (P = 0.199 gt; 0.05), the difference was not statistically significant, dose group compared to the total area of ??the neuronal cell bodies in the neuronal cell bodies of the total area and the blank control group (P = 0.000 lt; 0.05) difference was statistically significant; positive control group, the neuronal cell bodies of the total area and the blank control group of neuronal cell bodies total area compared (P = 0.000 lt; 0.05), the difference was statistically significant, prove that BDNF-TAT protect the biological activity of neuronal growth and survival in vitro. Conclusion 1. Of BDNF-TAT fusion protein through recombinant plasmid PET-30 (a)-BDNF-TAT expressed as inclusion bodies in E. coli BL21 (DE3) pLysS transformed to about a molecular weight of approximately 18kD. By the expression and purification of inclusion body proteins optimization and refolded to obtain high purity active BDNF-TAT fusion protein per liter of bacterial output target protein of approximately 4.8mmg purity of about 90%. 3 after tail vein injection renatured fusion protein BDNF-TAT by Western Blot analysis, the relative content of BDNF-TAT administered mice brain tissue compared with negative control group and blank control group was significantly high (P gt; 0.05) and the negative control group and blank control group, the relative content of BDNF-TAT was no difference (P lt; 0.05), prove that renatured fusion protein BDNF-TAT through the tail vein injection targeting peripheral to the brain tissue of mice. 4.SABC immunohistochemical method proved BDNF-TAT fusion protein after peripheral tail vein injection in mice brain tissue BDNF-TAT mainly distributed in the hippocampus CA1, CA3 and DG region. Vitro cultured dorsal root ganglion neurons that, BDNF-TAT fusion protein can promote the growth and survival of neurons.

Expression and Purification in E.coli and Activity in Vitro of Fusion Protein BDNF-TAT

Abstract

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