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Protein engineering and its techniques himanshu

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b pharma 6th sem
pharmaceutical biotechnology
Protein engineering
Objectives of protein engineering
Rationale of protein engineering
Protein engineering methods
Rational design -site-directed mutagenesis methods
Advantages and disadvantages of rational design
Directed evolution -random mutagenesis
Advantages and disadvantages of directed evolution
Peptidomimetics
Classification of peptidomimetics
Advantages and disadvantages of peptidomimetics
Flow cytometry
Instrumentation
Principle
components

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Protein engineering and its techniques himanshu

  1. 1. Protein Engineering 1 Himanshu Kamboj Assistant Professor
  2. 2. Contents  Protein engineering  Objectives of protein engineering  Rationale of protein engineering  Protein engineering methods  Rational design -site-directed mutagenesis methods  Advantages and disadvantages of rational design  Directed evolution -random mutagenesis  Advantages and disadvantages of directed evolution  Peptidomimetics  Classification of peptidomimetics  Advantages and disadvantages of peptidomimetics  Flow cytometry  Instrumentation  Principle  components
  3. 3. Protein Engineering Protein engineering can be defined as the modification of protein structure with recombinant DNA technology or chemical treatment to get a desirable function for better use in medicine, industry and agriculture.
  4. 4. OBJECTIVES OF PROTEIN ENGINEERING The objectives of protein engineering is as follows – (a) to create a superior enzyme to catalyze the production of high value specific chemicals. (b) to produce enzyme in large quantities. (c) to produce biological compounds(include synthetic peptide, storage protein, and synthetic drugs) superior to natural one.
  5. 5. RATIONAL OF PROTEIN ENGINEERING For industrial application an enzyme, should possess some characteristics in addition to those of enzymes in cells. These characteristics are :- (1) enzyme should be robust with long life. (2) enzyme should be able to use the substrate supplied in the industry even it differs from that in the cell. (3) enzyme should be able to work under conditions, e.g. extreme of pH, temperature and concentration of the industry even if they differ from those in the cell.
  6. 6.  In view of above, the enzyme should be engineered to meet the altered needs. Therefore efforts have been made to alter the properties of enzymes. These are some character that one might have to change in a predictable manner in protein engineering or enzyme engineering to get the desired function :-
  7. 7. Kinetic properties of enzyme-turnover and Michaelis constant, Km. Thermo stability and the optimum temperature for the enzyme. Stability and activity of enzyme in nonaqueous solvents. Substrate and reaction specificity. Cofactor requirements Optimum PH. Molecular weight and subunit structure.
  8. 8. Therefore for a particular class of enzymes, variation in nature may occur for each of the above properties, so that one may like to combine all the optimum properties to the most efficient form of the enzyme. For an e.g. glucose isomerases, which convert glucose into other isomers like fructose and are used to make high fructose corn syrup vital for soft drink industries.
  9. 9. Protein Engineering Obtain a protein with improved or new properties Proteins with Novel Properties Rational Protein Design Nature Random Mutagenesis 9
  10. 10. Protein engineering methods 1. Rational design 2. Site-directed mutagenesis 3. Evolutionary methods/directed evolution 4. Random mutagenesis 5. DNA shuffling 6. Molecular dynamics 7. Homology modeling 8. MolCraft‘in vitro protein evolution systems 9. Computational methods (computational protein design) 10. Receptor-based QSAR methods 11. X-ray crystallography 12. Peptidomimetics Phage display 13. Cell surface display technology 14. Flow cytometry / Cell sorting 15. Cell-free translation systems 16. Designed divergent evolution 17. Stimulus-responsive peptide systems 18. Mechanical engineering of elastomeric proteins 19. Engineering extracellular matrix variants 20.Traceless Staudinger ligation 21. De novo enzyme engineering mRNA display
  11. 11. Rational design • The most classical method in protein engineering is the so- called “rational design” approach which involves “site-directed mutagenesis” of proteins, to create improved protein molecules based on the three-dimensional structure and the relationship between structure and function. • Based on protein knowledge Structure Mechanism Dynamics Natural variation • Analogous to mechanical engineering
  12. 12. • Site-directed mutagenesis allows introduction of specific amino acids into a target gene. • There are two common methods for site-directed mutagenesis. • One is called the “overlap extension” method. This method involves two primer pairs, where one primer of each primer pair contains the mutant codon with a mismatched sequence. • These four primers are used in the first polymerase chain reaction (PCR), where two PCRs take place, and two double- stranded DNA products are obtained. • Upon denaturation and annealing of them, two heteroduplexes are formed, and each strand of the heteroduplex involves the desired mutagenic codon.
  13. 13. • The other site-directed mutagenesis method is called “whole plasmid single round PCR”. • This method forms the basis of the commercial “Quik Change Site-Directed Mutagenesis Kit” from Stratagene. • It requires two oligonucleotide primers with the desired mutation(s) which are complementary to the opposite strands of a double-stranded DNA plasmid template. • Using DNA polymerase PCR takes place, and both strands of the template are replicated without displacing the primers and a mutated plasmid is obtained with breaks that do not overlap. • DpnI methylase is then used for selective digestion to obtain a circular, nicked vector with the mutant gene. • Upon transformation of the nicked vector into competent cells, the nick in the DNA is repaired, and a circular, mutated plasmid is obtained
  14. 14. Site-directed mutagenesis methods 14
  15. 15. Site-directed mutagenesis methods – PCR based 15
  16. 16. Site-directed mutagenesis methods – Oligonucleotide - directed method 16
  17. 17. Advantages and disadvantages Advantages • Intellectually satisfying • Controlled outcome • Range of available techniques • Increasing computational power Disadvantages • Requires deep understanding  Natural variation  Structure  Dynamics  Mechanism • High failure rate Failures rarely reported
  18. 18. 18 Directed Evolution  This method mimics natural evolution and generally produce superior results to rational design  This also know as irrational design  An additional technique known as DNA shuffling mixes and matches the pieces of variants in order to produce better results.  In this random mutagenesis play an important role.
  19. 19. 19 Directed Evolution – Random mutagenesis - NO structural information required - NO understanding of the mechanism required General Procedure: Generation of genetic diversity  Random mutagenesis: when an organism exposed to physical or chemical mutagen, mutations are induced randomly in all genes of the organism. Hence, this process of generating mutations is known as Random mutagenesis. Identification of successful variants  Screening and seletion
  20. 20. 20 Directed Evolution - A simple and common technique for random mutagenesis is “saturation mutagenesis”. - It involves the replacement of a single amino acid within a protein with each of the natural amino acids, and provides all possible variations at that site. “ - Localized or region-specific random mutagenesis” is another technique which is a combination of rational and random approaches of protein engineering. - It includes the simultaneous replacement of a few amino acid residues in a specific region, to obtain proteins with new specificities.
  21. 21. 21 - This technique also makes use of overlap extension, and the whole- plasmid, single round PCR mutagenesis, as in the case of site- directed mutagenesis. - However, the major difference here is that the codons for the selected amino acids are randomized, such that a mixture of 64 different forward and 64 different reverse primers are used, based on a statistical mixture of four bases and three nucleotides in a randomized codon
  22. 22. 22
  23. 23. Random Mutagenesis (PCR based) with degenerated primers (saturation mutagenesis) 23
  24. 24. Random Mutagenesis (PCR based) with degenerated primers (saturation mutagenesis) 24
  25. 25. Random Mutagenesis (PCR based) Error –prone PCR -> PCR with low fidelity !!! Achieved by: - Increased Mg2+ concentration - Addition of Mn2+ - Not equal concentration of the four dNTPs - Use of dITP - Increasing amount of Taq polymerase (Polymerase with NO proof reading function) 25
  26. 26. Random Mutagenesis (PCR based) DNA Shuffling 26 DNase I treatment (Fragmentation, 10-50 bp, Mn2+) Reassembly (PCR without primers, Extension and Recombination) PCR amplification
  27. 27. Random Mutagenesis (PCR based) Family Shuffling Genes coming from the same gene family -> highly homologous -> Family shuffling 27
  28. 28. Advantages and disadvantages Advantages • Simple concepts • Widely applicable • High success rate • Requires no knowledge of protein (or of mutations) Disadvantages • Starting activity range Requires some starting activity Can rarely improve native activity • Requires high-throughput assay Typically enzyme-specific Screens can be expensive Synergistic mutations hard to find • Understanding results is not trivial: Especially to apply to another protein • You get what you screen for: Surrogate substrates can yield artefacts, Side- effects if not constrained
  29. 29. 29 Peptidomimetics  Peptidomimetics are compounds whose essential elements (pharmacophore) mimic a natural peptide or protein in 3D space and which retain the ability to interact with the biological target and produce the same biological effect.  It involves mimicking or blocking the activity of enzymes or natural peptides upon design and synthesis of peptide analogs that are metabolically stable.  Peptidomimetics is an important approach for bioorganic and medical chemistry.  It includes a variety of synthesis methods such as the use of a common intermediate, solid phase synthesis and combinatorial approaches.  Peptidomimetics are designed to avoid some of the problems associated with a natural peptide for example Stability against proteolysis (duration of activity) Poor bioavailability. Receptor selectivity or potency (often can be substantially improved)
  30. 30. 30 Peptidomimetics  The design process begins by developing structure– activity relationships (SAR) that can define a minimal active sequence or major pharmacophore elements, and identify the key residues that are responsible for the biological effect.  Then structural constraints are applied to probe the 3D arrangement(s) of these features. In this process, the peptide complexity is reduced and the basic pharmacophore model is defined by its critical structural features in 3D space.  This model then supports the re-assembly of the critical elements and non-peptide variants on a modified scaffold that presents the optimized pharmacophore to the receptor
  31. 31. 31 Classification of Peptidomimetics  Type-I peptidomimetics or pseudopeptides: These are synthesized by structure based drug design. These peptidomimetics are closely similar to peptide backbone while retaining functional groups that makes important contacts with binding sites of the receptors.  Type-II peptidomimetics or functional mimetics: These peptidomimetics are synthesized by molecular modeling and high throughput screening (HTS) etc. These are small non-peptide molecule that binds to a peptide receptor.
  32. 32. 32 Classification of Peptidomimetics  Type-III peptidomimetics or topographical mimetics: These are synthesized by structure based drug design which represents that they possess novel templates, which appear unrelated to the original peptides but contain the essential groups, positioned on a novel non- peptide scaffold to serve as topographical mimetics.  Type-IV Peptidomimetics or non-peptide mimetics: hese are synthesized by Group Replacement Assisted Binding (GRAB) technique of drug design.
  33. 33. 33 Advantages 1. Conformationally restrained structures can minimize binding to non-target receptors and enhance the activity at the desired receptor. 2. Addition of hydrophobic residues and/or replacement of amide bonds results in better transport properties through cellular membranes. 3. Isosteres, retro-inverso peptides, cyclic peptides and non-peptidomimetics all reduce the rate of degradation by peptidases and other enzymes.
  34. 34. 34 Disadvantages 1. Limited stability towards proteolysis by peptidases in the gastrointestinal tract and in serum (t1/2 on the order of minutes) 2. Poor transport properties from the intestine to the blood and across the blood-brain barrier due to high MW and lack of specific transport systems 3. Rapid excretion through the liver and/or kidneys 4. Inherent flexibility enables interaction with multiple receptors besides the target, and could result in undesired side-effects
  35. 35. 35 Flow cytometry Flow= flow Cyto= cell Metry= measurement “Flow cytometry”, a powerful method for single cell analysis, is also used in protein engineering studies. A variety of examples are available where the sorting was done according to ligand binding in antibody and peptide surface display studies, or enzyme engineering of intra- and extracellular enzymes.
  36. 36. 36 Flow cytometry The advantages and disadvantages of random mutagenesis methods used in protein engineering were also determined and compared to each other in detail. Based on the nucleotide substitution method used, these random mutagenesis methods were divided into four major groups: enzyme-based methods, synthetic chemistry-based methods, whole cell methods and combined methods. Their comparison was made according to a variety of parameters such as controllable mutation frequency, technical robustness, cost-effectiveness, etc
  37. 37. 37 HISTORY • Mark Fulwyler was the inventor of the forerunner of the modern flow cytometer. • This was developed in 1965 • First fluorescence based flow cytometer was developed in 1986by Wolfgang Gohde. • The flow cytometer was originally called pulse cytophotometry. • In 1988, the name was officially changed to “flow cytometry” at the Conference of the American Engineering Foundation in Pensacola, Florida.
  38. 38. 38 THE INSTRUMENT • Modern flow cytometers can analyze several thousand particles every second. • It can also actively separate and isolate particles having specified property.  A flow cytometry is composed of three main systems namely, fluidics, optics, electronics • Each cell is subjected to a laser beam. • The light reflected from each cell is captured. • Information is then interpreted statistically by a software
  39. 39. 39 COMPONENTS Liquid stream system: carries and aligns cells so that they pass through a single file. Measuring system: measure the impedance. optical system: lamps, high power and low power lasers Detector and analogue system computer
  40. 40. 40 PRINCIPLE o Beam of light of single wavelength is directed onto hydrodynamically focused stream of liquid o Detectors are placed where the stream passed through the light beam. o The light scattered by the cells are detected. o The amount of light scattered is measured. o This is analysed and results are interpreted.
  41. 41. 41 HYDRODYNAMIC FOCUSING Cells to be analyzed are suspended in liquid and forced through a small aperture. A tube is used through which the sheath fluid is pumped. Cells along with the fluid are forced through this narrow aperture. Cells move in a single file or line. Laser hits each cell and data from each cell can be read. SCATTERING Cells pass through the laser. Light gets refracted or scattered in all angles. 2 types of scatter are analyzed: • Forward scatter: scatter in the forward direction • Side scatter: light scattered in very large angles.
  42. 42. 42 FLOURESCENCE IN FLOW CYTOMETRY Most common method of studying cellular characteristics. Antibodies are tagged with a flourophore. The antibody binds to cells. When laser hits the flourophore, the molecule gets excited. Signal is emitted which can be detected. DETECTION OF FLOURESCENCE Fluorescent t light is travels the same path as side scatter. The light is directed through a series of filters and mirrors. Particular wavelength of light is detected by the appropriate detector.
  43. 43. 43 MEASURABLE PARAMETERS o Volume and morphological complexity of cells o Cell pigments such as chlorophyll o Cell cycle analysis, cell kinetics o Chromosomal analysis o Protein modification o Antigens o Enzymatic activity o Membrane fluidity
  44. 44. Thank you

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