Topic 2: Proteins. 2-1 specific proteins can be purified from cell extracts. Molecular Biology and Public Health ( 分子生物学与公共卫生 )

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1 HAPTER20: Techniques of Molecular Biology Molecular Biology and Public Health ( 分子生物学与公共卫生 ) Protein manipulation ( 蛋白操作 ) Topic 2: Proteins 1. Protein purification ( 蛋白质纯化 ) 2. Affinity chromatography can facilitate rapid protein purification ( 亲和层析纯化 ) 3. Protein separation by PAE gel electrophoresis ( 蛋白质分离 ) and identification by Western analysis 4. Protein sequencing ( 蛋白质测序 ) 5. Proteomics ( 蛋白质组学 ) specific can be purified from cell extracts The purification of individual is critical to understanding their function. (why?) Although there are thousands of in a single cell, each protein has unique properties that make its purification somewhat different from others. The purification of a protein is designed to exploit its unique characteristics, such as size, charge, shape, and in many instance, function.

2 2-2 purification of a protein requires a specific assay To purify a protein requires that you have an assay that is unique to that protein. In many instance, it s convenient to use a measure for the function of the protein, or you may use the antibody of the protein. It is useful to monitor the protein during the purification process. 2-3 Proteins can be separated from one another using column chromatography In this approach, protein fractions are passed though glass columns filled with appropriated modified small acrylamide or agarose beads. There are various ways columns can be used to separate according to their characteristics. Ion exchange chromatography Fig a The are separated according to their surface charge. The beads are modified with either negativecharged or positive-charged chemical groups. Proteins bind more strongly requires more salt to be eluted.

3 el filtration chromatography Fig b This technique separate the on the bases of size and shape. The beads for it have a variety of different sized pores throughout. Small can enter all of the pores, and take longer to elute; but large cannot enter pores and thus pass through column quickly. 2-4 affinity chromatography can facilitate more rapid protein purification If we firstly know that our target protein can specifically interact with something else, we can bind this something else to the column and only our target protein bind to the column. This method is called affinity chromatography. Protein structure Affinity chromatography Enzyme-substrate binding Receptor-ligand binding 12

4 Affinity chromatography Immunoaffinity chromatography An antibody that is specific for the target is attached to the bead, and ideally only the target protein can bind to the column. However, sometimes the binding is too tight to elute our target protein, unless it is denatured. But the denatured protein is useless unless we can renature it. 13 Sometimes tags (epitopes) can be added to the N- or - terminal of the protein, using molecular cloning method. This procedure allows the modified to be purified using immunoaffinity purification and a heterologous antibody to the tag. Importantly, the binding affinity can change according to the condition. e.g. the concentration of the a 2+ in the solution. immunoprecipitation We attach the antibody to the bead, and use it to precipitate a specific protein from a crude cell extract. It s a useful method to detect what or other molecules are associated with the target protein.

5 2-5 separation of on polyacrylamide gels The native have neither a uniform charge nor a uniform secondary structure. If we treat the protein with a strong detergent SDS, the higher structure is usually eliminated. And SDS confers the polypeptide chain a uniform negative charge. And sometimes mercaptoethanol is need to break the disulphide bond. Thus, the protein molecules can be resolved by electrophoresis in the presence of SDS according to the length of individual polypeptide. After electrophoresis, the can be visualized with a stain, such as oomassie brilliant blue. A protein gel stained by oomassie Blue Western analysis using two specific antibodies --- P+ P++ P+++ P++++ PTB Beta-actin 19 20

6 2-6 antibodies visualize electrophoretically-separated. The electrophoretically separated are transferred to a filter. And this filter is then incubate in a solution of an antibody to our interested protein. Finally, a chromogenic enzyme is used to visualized the filter-bound antibody. 2-7 protein molecules can be directly sequenced Two sequence method: Edman degradation & Tandem mass spectrometry(ms/ms). Due to the vast resource of complete or nearly complete genome, the determination of even a small stretch of protein sequence is sufficient to identify the gene. Edman degradation It s a chemical reaction in which the amino acid s residues are sequentially released for the N-terminus of a polypeptide chain. Step 1: modify the N-terminal amino with PIT, which can only react with the free α- amino group. Step 2: cleave off the N-terminal by acid treatment, but the rest of the polypeptide remains intact. Step 3: identify the released amino acids by High Performance Liquid hromatography (HPL). The whole process can be carried out in an automatic protein sequencer.

7 Tandem mass spectrometry MS is a method in which the mass of very small samples of a material can be determined. Fig Step 1: digest your target protein into short peptide. Step 2: subject the mixture of the peptide to MS, and each individual peptide will be separate. Step 3: capture the individual peptide and fragmented into all the component peptide. Step 4: determine the mass of each component peptide. Step 5: deconvolution of these data and the sequence will be revealed. Fig 20-24

8 2-8 proteomics Proteomics is concerned with the identification of the full set of produced by a cell or a tissue under a particular set of conditions. Three principle methods 1. 2-D gel electrophoresis for protein separation ( 蛋白质分离 ). 2. MS spectrometry for the precise determination of the molecular weight and identify of a protein ( 蛋白质鉴定 ). 3. Bioinformatics for assigning and peptides to the predicted products of protein coding sequence in the genome ( 蛋白质确定 ). 30 HAPTER20: Techniques of Molecular Biology Topic 3: Study the interaction between protein and nucleic acid 1. el retardation assay 2. Nuclease protection assay Fig

9 el retardation ( 凝胶阻滞 ) A short labeled nucleic acid is mixed with a cell or nuclear extract expected to contain the binding protein. Then, samples of labeled nucleic acid, with and without being incubated with the extract, are run on a native gel. The DNA-protein complexes are shown by the presence of slowly migrating bands. A DNA bound with more than one protein to form a larger complex. DNA bound to two DNA-protein complex Bare DNA 33 DNase I footprinting (DNase I 足迹法 ) Identify the actual region of sequence with which the protein interacts. Sequence ladder is required to determine the precise position Bind protein DNase(mild),then remove protein and denature DNA Dnase footprinting (1)The protein protects DNA from attack by DNase. (2) Treat the DNA-protein complex with DNase I under mild conditions, so that an average of only one cut occur per DNA molecule. 5 * AATAA 35 Electrophoresis, autoradiograph 36

10 HAPTER20: Techniques of Molecular Biology [Protein] T A A A A T T The three lanes represent DNA that was bound to 0, 1, and 5 units of protein. The lane with no protein shows a regular ladder of fragments. The lane with one unit shows some protection, and the lane with 5 units shows complete protection in the middle. By including sequencing ladders, we can tell exactly where the protein bound. 37 Topic 4: Determining the Structure of Protein and nucleic acids 1. X-ray crystallography (X- 晶体衍射 ) 2. NMR ( 核磁共振 ) 38 X-ray crystallography and NMR Determining the tertiary structure The ribosome structure and its interaction with mrna and trna X-ray crystallography: Measuring the pattern of diffraction of a beam of X-rays as it pass through a crystal. The first hand data obtained are electron density maps, the crystal structure is then deduced from electron density maps. A very powerful tool in understanding protein tertiary structure Many have been crystallized and analyzed

11 NMR Measuring the relaxation of protons after they have been excited by radio frequencies in a strong magnetic field. Measure protein structure in liquid but not in crystal. Protein measured are usually smaller than 30 KDa. 41 HAPTER20: Techniques of Molecular Biology Nucleic acids techniques: Electrophoresis; Restriction digestion; Hybridization (southern & northern); PR amplification; sequencing and genome sequencing; DNA cloning and gene expression. Protein techniques: Protein purification; affinity chromatography; Protein separation and identification by western blot; Protein sequencing; Proteomics. Study the interaction between protein and nucleic acid el retardation & Nuclease protection assays Determining the Structure of protein and 42 nucleic acids: X-ray crystallography, NMR Home Work 1. What does SDS do to the before and during SDS-PAE protein separation? 2. What characters of a protein can be used for its separation and purification? 3. Name two methods for studying nucleic acid-protein interaction. 43