Molecular Cell Biology. Prof. D. Karunagaran. Department of Biotechnology. Indian Institute of Technology Madras

Transcription

1 Molecular Cell Biology Prof. D. Karunagaran Department of Biotechnology Indian Institute of Technology Madras Module 5 Methods in Cell Biology (Methods to Manipulate Protein, DNA and RNA and Methods to Visualize Cells) Lecture 2 Methods to Manipulate Cells and Proteins

2 Isolating Cells Tissues contain a heterogeneous population of cells and these have to be isolated for biochemical studies. Disrupting extracellular matrix, cell-cell interactions and cell-cell adhesions is an important first step to isolate individual cells from tissues. This is done by treatment with proteolytic enzymes such as trypsin and with chelating agents such as ethylene diamine tetra acetic acid. If cells have to be multiplied in large numbers then they can be grown using standard cell culture protocols using appropriate media supplemented with serum and growth factors. Cultures of cells derived directly from tissues obtained from an organism are known as primary cultures. Plant cells can also be grown initially as a mass of undifferentiated cells called callus which can give rise to roots and shoots with appropriate media. Culturing cells Normal animal cells divide only for about times after which they do not divide further and this is called replicative cell senescence. Chromosomes get shortened after each cell division and short DNA sequences called telomeres are necessary to replenish this defect. Since normal cells lack telomerase that takes care of maintaining telomeres the cells are not able to divide forever. If telomerase is made active in these cells they can be grown continuously and are said to be immortalized However, not all human cells can be immortalized by this method of activating telomerase since they may have activated cell cycle checkpoint mechanisms that arrest the cell cycle. Such checkpoint mechanisms can be inactivated by introducing oncogenic viruses or oncogenes and thus the cells can be immortalized. Some rodent cells can inactivate these mechanisms and immortalized cells emerge spontaneously.

3 Immortalized cells prepared from cancer cells are called transformed cells and they can grow for several generations and do not need a solid support to grow. Such cell lines are useful in research and they can be frozen in liquid nitrogen at C and can be thawed again and they still retain their viability. Embryonic stem cells can also proliferate indefinitely and can develop into any other cell type. Specific cell types can be separated from a mixture of cells by fluorescence activated cell sorter. A specific cell type can be labeled with a fluorochrome coupled antibody that binds to its specific surface marker protein. The cells suspended in a fluid are passed through a narrow passage and come in contact with a laser beam and those which emit fluorescent light are now given a charge so that they can be deflected to fall into a collecting tube. Selected cells can be grown in culture or can be used for analysis. Fluorescence activated cell sorter

4 To analyze the biochemical components of cells, they are first subjected to sonication/osmotic shock/grinding. These methods open up the cells breaking them into pieces containing organelles, proteins and other components suspended in a homogenate or extract. The different components of this extract have to be separated by centrifugation. Cellular organelles such as nuclei, mitochondria, and lysosomes differ insize and specific gravity, and these properties are used in centrifugation to separate them. In differential centrifugation different components/organelles of a cell can be pelletted with different speeds of centrifugation. In isopycnic centrifugation, a centrifuge tube is filled with sucrose that is dissolved at different concentrations to produce a density gradient (lower concentration at the top and higher concentration at the bottom. When the cell extract is centrifuged at high speed, individual organelles sediment until their buoyant density exactly matches that in the gradient. Each layer can be collected separately. These methods are highly useful to study the functions of cell organelles/components in isolation. These fractions also serve as starting points for further purification of biomolecules in a cell.

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6 Purification of Proteins Cell extracts may be processed further with column chromatography to purify the proteins of interest. Column chromatography employs a glass or plastic cylinderical column filled with a permeable solid matrix such as cellulose. Cell extracts in suitable solvents are poured over the column and the various components of a cell are collected as various fractions at the bottom since they travel at different rates through the column.

7 In ion exchange chromatography, positively charged components can be retained in the column if an insoluble matrix that is negatively charged is used and vice versa. Examples include Diethyl aminoethyl cellulose (positively charged) and carboxymethyl cellulose and phosphocellulose (negatively charged) Ionic strength and ph of the solution can be changed systematically to achieve better separation and elution of the required proteins.

8 In gel filtration chromatography cross-linked beads of agarose, dextran or acrylamide are used as matrices. In this method small molecules penetrate into the matrices and stay inside for a long time before they emerge out of the column and hence the large molecules are separated out quickly. Affinity chromatography makes use of the affinity between an enzyme and substrate or a ligand and receptor or an antigen and antibody. The column is filled with matrix that is covalently linked to an enzyme/antibody/ligand. The elution is based on differences in ph or salt concentrations, the conditions in which the affinity can be changed drastically to liberate the binding partners. Silica-based resins are used in high performance liquid chromatography that achieves greater resolution to separate proteins.

9 SDS Polyacrylamide Gel Electrophoresis Proteins have a net positive or negative charge depending on their amino acid composition. Electrophoresis is a popular technique that applies electric field to separate proteins based on their net charge, size and shape. Sodiumdodecyl sulfate (SDS) is a detergent that can give a net negative charge to most of the different types of proteins and thus proteins behave as anions and move towards anode when an electric field is applied. SDS gel electrophoresis uses polyacrylamide gels that are highly cross linked and the consequent pores act as sieves for different proteins and separate them depending on their molecular size apart from the net charge. The disulfide bridges in proteins are broken by the addition of reducing agents such as β-mercaptoethanol and this makes the subunits to separate out and migrate in the gel.

10 The proteins migrate in the gel depending on their size and can be visualized by staining with dyes such as Coomassie Blue. SDS polyacrylamide gel electrophoresis is very useful to separate the subunits of proteins and to assess their molecular weights. Proteins thus separated are usually transferred to a nitrocellulose membrane and then identified using specific antibodies coupled with radioactive/fluorescent/luminescent secondary reagents. This method of transferring the proteins on to a membrane and subsequent detection with labeled antibodies is called Western blotting/immunoblotting. Two-dimensional Gel Electrophoresis Basically the technique separates proteins by electrophoresis in two dimensions. The protein sample is first solubilized with a nonionic detergent together with urea and β-mercaptoethanol to keep their intrinsic charge unchanged. In the first dimension the proteins are separated by isoelectric focusing in a narrow tube of polyacrylamide gel that contains a ph gradient by adding a mixture of special buffers. Proteins have their own isoelectric ph (at this ph there is no net charge) and at this ph in the gradient gel they do not move, thus helping in their separation. The separated proteins are now run in the second dimension using SDSpolyacrylamide gel electrophoresis at right angles to the direction in which isoelectric focusing was carried out. The proteins can then be visualized by various staining techniques In this manner protein having same isoelectric ph can also be separated in the second dimension.

11 Mass spectrometry Proteins from a number of different living organisms have been identified and catalogued and this information can be used to identify an unknown proteins isolated from an organism/cell extract Proteins are first digested to produce smaller peptides and then a laser beam is directed towards the peptides that can be ionized in a closed chamber under vacuum. Under an electric field such ionized peptides will move towards an electrode of opposite charge depending on their net charge and the time taken to reach the electrode gives information about the mass of the peptide. Using a database that contains information about proteins and their peptide fragments under these conditions, the unknown peptide can be precisely identified in most cases. This technique is called matrix-assisted laser desorption time of flight (MALDI- TOF) spectrometry. MALDI-TOF MALDI-TOF can be used to determine the molecular weights of proteins and peptides. As an improvement of MALDI-TOF, collisions with high-energy gas atoms can be used to preferentially cleave the peptide bonds, generating a ladder of fragments, each differing by a single amino acid. A second mass spectrometer can separate these fragments and display their masses. The amino acid sequence of a peptide can then be deduced from these differences in mass and this technique is called MS/MS and can be used to detect post translational modifications of proteins such as phosphorylation/acetylation that change the mass of the protein. Another technique called LC-MS/MS digests the proteins with trypsin first and the short peptides produced are subjected to a series of automated liquid chromatography steps. As a second dimension the separated peptides are analyzed by MS/MS technique.

12 X-Ray Diffraction Three dimensional structures of proteins have been determined mainly by using X-ray diffraction. X-rays are electromagnetic radiations of short wavelength (0.1 nm) A narrow beam of X-rays can be directed on a protein crystal and the scattered rays get reinforced and appear as a diffraction pattern detectable by suitable detectors. The diffraction pattern can give rise to an electron density map that has to be carefully interpreted to get the three dimensional structure of a protein. Proteins are needed in a large quantity and good crystals have to be obtained for this technique to be effective Nuclear Magnetic Resonance (NMR) Not all proteins are crystallized in the laboratory efficiently and hence this technique is quite useful to determine the structures of proteins without crystals as it only requires a concentrated solution of proteins for structural determination. NMR measures the absorbance of radio frequency electromagnetic energy by certain atomic nuclei.

13 This technique depends on the fact that certain atomic nuclei are intrinsically magnetic. Only a limited number of isotopes display this property, called spin. The spinning of a proton generates a magnetic moment. This moment can take either of two orientations, or spin states (called α and β) when an external magnetic field is applied. The energy difference between these states is proportional to the strength of the imposed magnetic field. The α state has a slightly lower energy because it is aligned with this applied field. A spinning proton in an α state can be raised to an excited state (β state) by applying a pulse of electromagnetic radiation (a radio-frequency, or RF, pulse), provided that the frequency corresponds to the energy difference between the α and the β states. In these circumstances, the spin will change from α to β; in other words, resonance will be obtained. Basis of NMR microscopy

14 Study Questions 1. How are proteins purified from cell extracts? 2. How does a typical cell sorter work and what are its applications? 3. Cells can be fixed with a) nitric acid b) Glutaraldehyde c) ethanol d) paraffin 4. Match the following NMR X-ray Diffraction SDS-PAGE Iso electric focusing ph Molecular weight Electron spin Electron scattering 5. For proteins that cannot be crystallized method can be used for obtaining structural information.