Protein expression Analysis and its applications Proteins: An Introduction Amino acids covalently bonded successively to form complex macromolecules called as proteins. Every protein has a unique sequence of amino acids that linked together through peptide bonds to form linear structures. Moreover, proteins have four levels of structures that are: • Primary Structure (linear arrangement of proteins) • Secondary structure (hydrogen bonds formed, a- helices and ß-structures formed) • Tertiary structure (formed by intramolecular arrangement of secondary structure) • Quaternary structure (formed by stichometry and spatial arrangement of protein subunits) After the sequencing it reveals that human genome comprised up of 30,000 to 40,000 genes and proteins that are encoded by theses gene is three to four times higher. Complexity of organism depends on its proteome and thus it is important to study proteins and protein’s expression.i Protein Expression Protein expression is named to the process of formation of protein and then its regulation inside the cell. Proteins are essential for the body and are required for a lot of processes. Proteins are formed inside the cell by a process known as central dogma. First, the transcription take place in which gene is transcribed and mRNA is formed. This mRNA is processed and subjected to another phenomenon called as splicing in which introns get spliced from mRNA and exons are joined again by ligases. The coding regions are exons which are then transported to cytoplasm form nucleus and translation is done. During translation mRNA sequence is converted in to protein which is made up of specific sequences of amino acid. Figure 1Protein formation Recombinant proteins can also be formed, for the large-scale production. In this gene of specific protein is inserted in bacterial genome and hence proteins are formed, isolated and analyzed.ii Protein Expression Analysis: Proteins vary from each other in the type, number and sequence of amino acids that make up the polypeptide backbone of the specific protein. So, proteins have different molecular structure, function, and properties. Protein expression analysis means the isolation, identification, quantification and qualification of a specific protein. Protein expression analysis can be done by different ways which are as follows: • separation techniques • antibody specific techniques • spectrometric techniques Many techniques are used to carry out these basic steps, details of them are discussing below. Protein Analysis by Separation Techniques Proteins when formed they are intracellular, extracellular or periplasmic proteins. First, they get isolated from the cells by centrifugation. A mixture of proteins, RNA and DNA is obtained. Proteins are purified from the mixture leaving RNA and DNA. The mixture of proteins then processed by using different techniques to get separated the protein of interest and during this separation proteins get identified. Different methods of protein analysis by separation method are: • SDS-PAGE • Isoelectric Focusing • 2-D Electrophoresis SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis is a technique used to separate proteins from a mixture due to difference in their sizes. This technique is based on the principle that the charged molecules moved towards opposite electrode in an electric field. So, proteins get linearized by using SDS and then they become negatively charged as well. In presence of electric charge, they move towards the positive electrode and get separated on basis of size. By knowing the sizes of proteins, the protein of interest is identified and further confirmed as well. Larger protein covers less distance in gel and smaller proteins covers large distance. iii Isoelectric Focusing Isoelectric focusing is a method used to separate and identified proteins on basis of pH. This electrophoretic method uses the property of proteins that the charge on protein can be determined by the pH of the environment. Proteins are either neutral, positively or negatively charged and the charge of protein also depends upon the type of amino acids. Proteins get positively charged at pH below their isoelectric point and move towards cathode and get negatively charged in pH above their pI and moves towards anode so on this basis they get separated. iv Figure 2 Showing Isoelectric Focusing of Proteins 2-D Electrophoresis Two-dimensional gel electrophoresis (2DE) is the method used to isolate and identify proteins based on their charge (isoelectric focusing, IEF) and of their size (sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE). In 2-D electrophoresis proteins get separated in two dimensions. In first dimension proteins are separated on basis of their isoelectric point and in second dimension further separation is carried out because of their molecular weight. v Figure 3: 2-D Electrophoresis Protein Analysis by Antibody Specific Techniques Protein analysis can also be done by using antibody methods. Protein specific antibodies are used for the detection of the specific protein. Techniques that falls udder this are as follows: • Immunoassays • ELISA • Western Blot Immunoassays Immunoassays are the identification techniques depending upon the target specific antibody. Immunoassay worked on the basic principle which is the binding between target and the target specific antibody. This is also called as “binding-assays”. Immunoassays are widely used for protein expression analysis. Immunoassay techniques are based on a competitive binding reaction between a fixed amount of labelled form of an analyte and a variable amount of unlabeled sample analyte for a limited quantity of binding sites on a highly specific anti-analyte antibody. When these immunoanalytical reagents are mixed and incubated, the analyte is bound to the antibody forming an immune complex. This complex is separated from the unbound reagent fraction by physical or chemical separation technique. Analysis is achieved by measuring the label activity (e.g. radiation, fluorescence, or enzyme) in either of the bound or free fraction. A standard curve, which represents the measured signal as a function of the concentration of the unlabeled analyte in the sample is constructed. Unknown analyte concentration is determined from this calibration curve.vi ELISA ELISA stands for Enzyme-linked immunosorbent assay is an immunoassay used to identify proteins. In this technique solid phase enzyme immunoassay is used for the detection of substance specifically protein. Proteins get attached to the surface, normally a microtiter plate and specific antibody is applied to that protein and binding occurs. Enzyme is also linked to antibody and enzyme substrate is added and color is change due to reaction and protein identification is done. vii Western Blot Western blotting (protein blotting or immunoblotting) is a rapid and sensitive assay for detection and characterization of proteins. It is based on the principle of immunochromatography where proteins are separated into polyacrylamide gel according to their molecular weight. It is an analytical method wherein a protein sample is electrophoresed on an SDS-PAGE and electro transferred onto nitrocellulose membrane. The transferred protein is detected using specific primary antibody and secondary enzyme labeled antibody and substrate. A protein sample is subjected to polyacrylamide gel electrophoresis. After this the gel is placed over a sheet of nitrocellulose and the protein in the gel is electro phonetically transferred to the nitrocellulose. The nitrocellulose is then incubated with the primary antibody for the protein. The nitrocellulose is then incubated with a secondary antibody, which is specific for the first antibody. The second antibody will typically have a covalently attached enzyme which, when provided with a chromogenic substrate, will cause a color reaction.viii Figure 4: Protein Analysis by Western Blot Protein analysis by spectrometric methods Spectrometric methods are used for the detection of proteins. In such methods the amount of light or radiation or energy emitted by sample is analyzed and its identification is done. Spectrometric methods used for protein analysis is Mass Spectrometry HPLC HPLC is a form of liquid chromatography, which is used to separate and purify proteins, where separation is carried out among a mobile and stationary phase. In HPLC separation is carried out under the same principle, separation of a sample into its component parts due to variance in the relative affinities of different molecules of proteins for the mobile phase and the stationary phase used in the separation. Proteins are injected in the column and then the proteins are detected by detector.ix Figure 5: showing Schematic Diagram of HPLC Mass spectrometry Mass spectrometry is separation technique that is used to separate unknown components from known compounds within a sample, predicts the structure and chemical properties of different molecules. In this process, the sample is converted into gaseous ions and they are separated on the basis of the mass to charge ratios (m/z) and relative abundances. Principally, this technique is based on the ionizing energy on molecules. It creates multiple ions from the sample and according to the specific mass-to-charge ratio (m/z) are seperated and the relative abundance of every single ion is being recorded. In the mass spectrometric analysis of compounds, the first step is the production of gas phase ions of the compound, mostly done by electron ionization. Every molecular ion undergoes the fragmentation process and thus seperated on their mass to charge ratios, and their respective abudances can be recorded. By using these ratios and relative abundances, a mass spectrum is produced in which ions provide information regarding structure of the specific molecule. In the spectrum of a pure compound, the molecular ion is present at the highest value of m/z (followed by ions containing heavier isotopes) and depicts the molecular mass of the compound.x Applications Protein expression analysis techniques have many applications which are listed below: • SDS-PAGE is used for the detection of proteins and other small molecules in measuring molecular weight, peptide mapping, estimation of protein size, estimation of protein purity, protein quantitation and analysis of the number and size of polypeptide subunits. • Isoelectric focusing is used to determine the size of protein, used to separate proteins and net charge of protein. It also used in the forensics and determination of food and plants proteins. xixii • 2-D Electrophoresis has its applications in studying proteomics of various diseases like breast cancer, and the proteome of crops of different kinds. xiiixiv • ELSIA is used in the testing of different viruses, in the detection of proteins, in the identification of different diseases. Used in food industries to check out the food hygienic quality and for the detection of different allergies.xv • Western Bolt is used in detection of proteins from different sources, also used in food industries, forensics and in medical testing of different viruses’ ad viral proteins. • HPLC has it applications in pharmaceutical industries for the purification of proteins, in food industries, medicines, in food analysis and in elevation of phytoconstituents. xvi • Mass spectrometry is used in many fields including, forensic toxicology, metabolomics, proteomics, pharma/biopharma, and clinical research. Specific applications of mass spectrometry include drug testing and discovery, food contamination detection, pesticide residue analysis, isotope ratio determination, protein identification, and carbon dating. Conclusion Protein expression analysis is important in knowing the internal composition of protein, its amino acid composition, structures, its subunits etc. Protein expression analysis is important as after the expression of recombinant protein its is necessary task to find out weather the protection is accurate or not. Many techniques are used for purification, specific and non-specific detection of proteins. These techniques give us confirmation of protein on different levels, either by confirming its size, its subunits etc. So, protein expression analysis is necessary to find out the protein of interest.