Expression, Purification And Crystallographic Studies Of Thermophilic Polygalacturonase CbPelA And HIV-1 Vif Mediated E3 Ubiquitin Enzyme Complexes

The first part: Expression, purification and crystal structure of thermophilic polygalacturonase from Caldicellulosiruptor bescii DSM6725Galacturonic acid by alpha 1,4 glycosidic bond polymerized polysaccharide chains, were called the pectic substances. A series of the floorboard of the enzyme hydrolyzed pectic substances as the substrate, known as pectinase. Polymer can be pectin polysaccharide effective degradation of reducing sugar in the form of small molecular fragments or monomer, distributed widely in higher plants and microorganisms. From decomposition methods, Pectinasecan can be divided into two categories:pectin hydrolases and pectin lyase. The former mainly included the original pectinase, pectinlipase, low poly-galacturonide glycanohydrase, methylgalacturonase and polygalacturonase; The latter mainly included polygalacturonlyase and methylpolgalacturonlyase. Hydrolytic enzyme hydrolyzed glycosidic bond by introducing water molecules, and lyase were through trans elimination effect, fractured glycosidic bond. Pectinase was a hotspot of current research industry, they can be applied to various industries, mostly in juice production and in papermaking industry a. In recent years, pectinase gradually began to used in brewing, feed manufacturing, textile industries.The paper studied a novel thermophilic polygalacturonase which were belonged to glycoside hydrolases 28 family. The gene of the polygalacturonase was amplified by PCR(Polymerase Chain Reaction) from the Caldicellulosiruptor bescii DSM6725 and subcloned into the vector pET-20b(+). The constructed recombinant plasmid was transformed to competent Escherichia coli BL21 (DE3) Codon plus and induced with IPTG. Recombinant protein CbPelA have been characterized, its optimum temperature was 72℃ respectively, the optimum pH was 5.2, and CbPelA maximum reaction rate was 386.8 U/mg -1, it had a good thermal stability and a half-life for 14 h respectively, thin-layer chromatography and decline in coefficient of viscosity experiments confirmed that the enzyme belonged to excision enzymes, the pectin enzyme activity for galacturonic acid polymer was very specific, for the CMC, Avicel, glucan, xylan, chitosan, soluble starch and other polysaccharide were no vitality. Monovalent ions Na+, K+, NH4+ were small effects on enzymatic reaction; but Most of divalent metal ions Mg2+、Zn2+、Ca2+、Ba2+、Co2+ and Ni2+ inhibited enzymatic reaction in different levels, including Cu2+ and Cd2+ completely inhibited CbPelA enzyme activity. In order to further study the relationship between the structure and function of protein, such as active center, the substrates, the substrate specificity and thermal stability, we studied the crystallography of CbPelA.In this paper, after the recombinant expression CbPelA target protein, the expressed recombinant protein was purified by affinity chromatography, ion-exchange chromatography and molecular sieve chromatography. The purified protein was screened with crystal kits. Gel electrophoresis analysis indicated that the gene of about1300bp was amplified with the annealing temperature at 58oC by PCR. For screening the positive recombinant plasmid in cloning sequencing results showed that they were consistent with those we successfully won the genome database of galacturonic acid polymer enzyme gene sequence; DNAsequencing proved that the recombinant plasmid was constructed correctly. The purified recombinant protein reached a purity of about 95% and 10 mg/L. Further we used Crystal Screen HR2-110 and Crystal Crystal Screen 2 HR2-112 kits for preliminary screening, microscope found that the Crystal Screen HR2-110 26 number:0.2 M Ammonium acetate,0.1 M citric acid ammonium acetate three sodium dihydrate pH5.6,30% 2-methyl-2,4-butyl glycol.In the medium, we found a small amount of protein Crystal, these had good transparency. Protein crystal was continued optimized, under the condition of other condition is constant, we changed the pH and precipitation dose respectively in the screening, we cultured observation was conducted in the 16℃, the part of the work is in progress.The second part: Expression, purification and crystallographic studies of HIV-1 Vif mediated E3 ubiquitin enzyme complexesHuman immunodeficiency virus (HIV)-1 virion infectivity factor(Vif) protain is one of six accessory protein encoding virion. The molecular weight of it is about twenty three kD. It widely exists in all the slow virus in the family retrovirus. In different types of cells, the protein Vif is based on the influence of the virus infection has great difference. In non-permissive cell, the lack strains of Vif can be normally grow and secrete, but can’t effectively copy, it will produce no infectious virus, which cannot be completed the next round of infected cells. However, in permissive cells, viral replication does not depend on Vif, in other words, HIV-1 virus with genetic defected Vif can also be growth and efficient replication in these cells, producing infectious virus.So, in non-permissive cell, the Vif protein of HIV-1 plays an important rolein resistance to host innate immune, it through ubiquitin protease degradation pathways in the cell to limit the antiviral activity of limiting factors of the host. Mainly in the human body cells express restrictive factors include AP0BEC3 family members, they can stop the replication and spreading of HIV. Their main role in the early life cycle, the virus can pack into a variety of retrovirus (such as HIV-1-2/HIV, equine infectious anemia virus, a variety of simian immunodeficiency virus, etc.), then start mass for inverse virus negative righteousness new chain cDNA deamination, high to trigger a viral genomemutations, cause viral protein missense mutation or early termination viral protein transcription.The mechanism mainly through the form with six conjoined E3 ligase complex that Vif is based as joint proteins, including CBF-β, cullin5, elonginC, elonginB, RBX protein, after E3 cascade will ubiquitin A3G, and by proteasome pathway in the cell A3G will bedegraded. As a joint protein, Vif and E3 complex function site has been studied more thoroughly, the sites of E3 components interact with Vif include: Vif amino end with CBF-β, Vif carboxyl end SOCS motif of conservative sites with elonginC SLQ, Vif is based SOCS motif downstream conservative HCCH motif and cullin5 union. Including cullin 5 protein as a skeleton protein, EB-EC as regulatory subunits, CBF-β as the complementary factor of Vif protein folding, together form the E3 ligase of reactivecomplex. However, the structure and function of relationship of HIV-Vif has yet to be clear.In order to understand the interaction mechanism between them, we built Vif(1-176)-CBF-β(1-165)-elonginB(1-118)-elonginC(12-112)-cullin5(1-386)-APOBE C3H(1-183) complex. We connected each purpose fragment with different carrier:Vif gene inserted pET21a carrier, CBF-β gene inserted pRSF carrier, ELOB-ELOC gene fragment inserted pCDFS common carrier, CUL5 gene insertedpET28a carrier, A3H gene inserted pET28a carrier. After the success of the recombinant plasmid construction, pET21a-Vif, pRSF-CBF-β and pCDFS-ELOB-ELOC were transferred into E.coli expression strain BL21, while pET28a CUL5 and pET28a A3H respectively into E.coli expression strain BL21. The application of affinity, ion exchange and molecular sieve chromatography method purified thecompound protein. The gel electrophoresis showed that under the condition of PCR 54℃ annealing temperature can be successfully get their purpose proteingene fragment by gene amplification. For screening the positive recombinant plasmid in cloning, the sequencing results show that we successfully obtained gene sequences consistented with genome database. Firstly recombinant proteins purified by Ni-NTA column respectively, four yuan, CUL5 and A3H protein purity of 90% can be obtained respectively, these concentration were about 10 mg/mL. Due to we found that A3H protein were easy to precipitate, so after Ni-NTA column purification, we mixed six kinds of protein, purified together by Resouce Q, Superdex 200 column, immediately. Molecular sieve results showed that the molecular weight is bigger than the size of the protein molecular weight in theory, there might be the purpose protein presented in the form of aggregation, there might be some embedded nucleic acid. Finally through SDS-PAGE gel electrophoresis showed that we can clearly saw strips of six yuan and we can obtained stable compound protein of six yuan. Finally We successfuliy obtained Vif-CBF-β-ELOB-ELOC-CUL5-A3H six yuan compound protein, laid the research foundation of its analysis of protein structure and function relationship, extended understanding of Vif-CUL5 E3 compound function mechanism, provided new possibilities for HIV medications.