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旺季：7月1日-次年春节

海戴的金牌编辑Catherine Du所撰写的分子和结构生物学研究计划范文（客户已经成功申请德国名校博士学位入学）

Structural and Functional Studies of Human Sun1

OBJECTIVES

•  To determine the orientation of Sun 1 in biological membranes.

•  To express human Sun 1 in E. coli.

•  To purify and refold the recombinant protein .

•  To study human Sun 1' s 3D structure and molecular interactions by NMR or X-ray crystallography.

OVERVIEW OF THE STUDY

Human Sun 1' s 3D structure and its interaction with other components in the nuclei are unknown. Recombinant proteins are popular choices in studying such issues. We plan to express human Sun 1 in a suitable Escherichia coli expression system which has several advantages over other expression systems, such as no post-translational modifications like glycosylation. By different approaches, such as mutant selection, protein fusion, using protease-deficient strains, etc., we can overcome toxicity and proteolysis which are often associated with expression of membrane proteins . If human Sun1 is expressed directly in the E. coli membrane, we could obtain valuable membrane models to study Sun 1' s orientation and in vivo interactions with other molecules. Next we will purify the recombinant protein by affinity chromatography which is one of the two suitable methods to study membrane proteins.

After the purification, we will refold the protein with the aid of micelles or liposomes in order to prepare samples for NMR study. S tudies have shown that Sun1 is detergent, salt, and chaotrope resistant . Fortunately, it is also shown that treatment with detergent and medium salt concentrations (1% Triton X-100/ 200 mM NaCl) can efficiently solubilize Sun1, which can be a good start for our study.

Currently, membrane proteins can be studied either by NMR or X-ray crystallography. However, for NMR, only protein fragments with molecular weight less than 20 kDa can be studied. For the limitation of NMR, we can study independently the transmembrane domains of Sun1 by NMR, and combine the experimental data, which can sometimes give sufficient information about the target protein. EXPERIMENTAL PROCEDURE

•  Sun1 polyclonal antibodies

Synthesize a peptide having the first 14 amino acid residues of human Sun1 (MDFSRLHMYSPPQC; NP_079430 ). Sun1 polyclonal antibodies will be produced against this peptide in rabbit. The anti-Sun1 serum IgG fraction (referred as the Sun1 serum) will be purified on a protein A-Sepharose column.

2. Recombinant Proteins

Sun1 was initially isolated from human brain. SUN will be PCR-amplified from the Sun1 cDNA and directly cloned in pcDNA3.1 TOPO/V5-His and pET21 vectors. Recombinant pET vectors will be transformed into BL21(DE3)plysS host which is ideally stringent to express membrane proteins. Verify if recombinant protein is expressed in inclusion bodies and purify it by Ni column.

3. Gel electrophoresis and Immunoblotting

Test the dilution first and then use the Sun1 serum as the primary antibody.

4. Proteinase K Protection Assays

The Sun1 cDNA cloned in the pcDNA3.1/V5-His vector will be used to synthesize the full-length protein in vitro with [ 35 S]methionine in the absence or the presence of canine-pancreatic microsomal membranes. Aliquots of the synthesis reaction will be digested for 30 min on ice with proteinase K. Proteinase K will be inhibited with PMSF, and the samples will be added directly to boiling Laemmli buffer. Duplicate samples will be transferred to a nitrocellulose membrane, and the radiolabeled proteins will be visualized with a PhosphorImager. The same membranes then will be immunoblotted with either the anti-Sun1 serum or the anti-V5 antibody.

5. Reconstitute Recombinant Protein Fragment Functionally into Detergent Micelles

In order to prepare samples for NMR study, we will first choose suitable detergent micelles to reconstitute the recombinant protein fragment , use a direct dilution method to refold the proteins, and test the refolding of the target protein by radio-ligand binding.

6. NMR and X-ray Crystallography Studies

We will examine whether solution NMR studies of the micelle systems ( using different domains of the target protein ) will give sufficient information about the structures of human Sun1. As for X-ray crystallography studies of the whole protein , we will choose a suitable detergent first to work out protein crystals and then perform structural studies on the crystals .

FURTHER STUDIES

•  With the aid of NMR and X-ray crystallography, we can study directly protein-drug interactions and protein-protein interactions.

•  We will predict human Sun 1' s 3D structure by molecular modeling and compare it with experimental data from NMR or X-ray crystallography.

郑重声明：此研究计划为Catherine Du为国内一知名留学文书公司所撰写的原创作品，未经允许，严禁传统媒体和网络转载，否则将追究法律责任。