Kareem Pharma Biotech Topic 2 PDF

Course contents Pharmaceuticals, biologics and biopharmaceuticals CONCEPTS AND METHODS FOR RECOMBINANT DRUG PRODUCTION Protein structure Gene manipulation and recombinant DNA technology The drug development process Sources of biopharmaceuticals & upstream processing  Procaryotic Ce...

Course contents Pharmaceuticals, biologics and biopharmaceuticals CONCEPTS AND METHODS FOR RECOMBINANT DRUG PRODUCTION Protein structure Gene manipulation and recombinant DNA technology The drug development process Sources of biopharmaceuticals & upstream processing  Procaryotic Cells, mammalian Cells ,Plants, insects, and transgenic Animals BRINGING THE DRUG INTO ACTION Downstream processing Product analysis RECENT APPLICATIONS IN PHARMACEUTICAL BIOTECHNOLOGY Metabolic Engineering of Medicinal Plants Metabolomics as Bioanalytical Tool Nutraceuticals/Functional Foods for Improving Health and Preventing Disease CONCEPTS AND METHODS FOR RECOMBINANT DRUG PRODUCTION Protein structure  Almost all products of modern pharmaceutical biotechnology are protein based. So, understanding of protein structure is central to this topic. Overview of protein structure Proteins are macromolecules consisting of one or more polypeptides. Each polypeptide consists of a chain of amino acids linked together by peptide (amide) bonds. The exact amino acid sequence is determined by the gene coding for that specific polypeptide. When synthesized, a polypeptide chain  A protein’s structure currently cannot be predicted simply from its amino acid sequence. Its conformation can, however, be determined by techniques such as X-ray diffraction (X-ray crystallography) and nuclear magnetic resonance spectroscopy (NMR). Proteins are sometimes classified as:  Simple proteins, consist exclusively of polypeptide chain(s) with no additional  Conjugated proteins, in addition to their polypeptide components(s), contain one or more non-polypeptide constituents known as prosthetic group(s). The most common prosthetic groups found in association with proteins include carbohydrates (glycoproteins), phosphate groups (phosphoproteins), vitamin derivatives (e.g. flavoproteins) and metal ions (metalloproteins). Examples of proteins The 20 commonly occurring amino acids One Example Amino acid: Basic unit of protein Carboxylic acid group COO- NH3 + C H Different side chains, R, determine the Amino group properties of 20 R amino acids (Neutral, acid, base). An amino acid The chemical structure of the 20 amino acids commonly found in protein  20 Amino acids Glycine Alanine (A) Valine Isoleucine (I) Leucine (L) (G) (V) Proline (P) Methionine (M) Phenylalanine (F) Tryptophan (W) Asparagine (N) Glutamine (Q) Serine Threonine (T) Tyrosine (Y) Cysteine (C) (S) Asparatic acid (D)Glutamic acid (E) Lysine (K) Arginine (R) Histidine (H) White: Hydrophobic, Green: Hydrophilic, Red: Acidic, Blue: Basic Each protein has a unique structure! Amino acid sequence NLKTEWPELVGKSV EEAKKVILQDKPEAQ IIVLPVGTIVTMEYRI DRVRLFVDKLD Folding!  Protein Structure STRUCTURE Primary Assembly PROCESS Secondary Folding Tertiary Packing Quaternary Interaction  Protein Assembly occurs at the ribosome involves polymerization of amino acids attached to tRNA yields primary structure Primary structure The primary structure : refers to the exact amino acid sequence, along with the exact positioning of any disulfide (-S-S-) bonds present Amino acid sequence determination The amino acid sequence of a polypeptide may be determined directly via chemical sequencing or by physical fragmentation and analysis, usually by mass spectrometry. Direct chemical sequencing was the only method available until the 1970s. Insulin was the first protein to be sequenced by this approach (in 1953), requiring several years and several hundred grams of protein to complete. The method has been automated over the years, such that, today, polypeptides containing 100 amino acids or more can be automatically sequenced within a few hours, using microgram to milligram levels of protein. Amino acid sequence is encoded by DNA base sequence in a gene DNA ・ ・ DNA base G C molecule C G sequence G C C G T A ＝ T A A T A T G C C G G C C G ・ ・ Amino acid sequence is encoded by DNA base sequence in a gene Second letter T C A G TTT TCT TAT TGT T Phe Tyr Cys TTC TCC TAC TGC C T TTA TCA Ser TAA TGA Stop A Leu Stop F TTG TCG TAG TGG Trp G T CTT CCT CAT CGT T ir His h CTC CCC CAC CGC C s C Leu Pro Arg ir CTA CCA CAA CGA A t CTG CCG CAG Gln CGG G d l ATT ACT AAT AGT T l e Asn Ser e ATC Ile ACC AAC AGC C tt A ATA ACA Thr AAA AGA A tt e Lys Arg e ATG Met ACG AAG AGG G r GTT GCT GAT GGT T r Asp GTC GCC GAC GGC C G GTA Val GCA Ala GAA GGA Gly A Glu GTG GCG GAG GGG G The major primary sequence databases (protein and nucleic acid)  Protein Structure STRUCTURE Primary Assembly PROCESS Secondary Folding Tertiary Packing Quaternary Interaction  Protein Folding occurs in the cytosol involves localized spatial interaction among primary structure elements, i.e. the amino acids. yields secondary structure The structure and function of a protein depends on protein folding If protein is folded incorrectly, desired function of a protein is lost and a misfolded protein can be detrimental  Secondary Structure non-linear 3 dimensional localized to regions of an amino acid chain formed and stabilized by hydrogen bonding Secondary Structure α-helix β-sheet Secondary structures, α-helix and β-sheet, have regular hydrogen-bonding patterns.  Protein Packing occurs in the cytosol involves interaction between secondary structure elements and solvent yields tertiary structure  Tertiary Structure non-linear 3 dimensional Tertiary structure refers to the overall folding of the entire polypeptide chain into a specific 3D shape  Protein Structure STRUCTURE Primary Assembly PROCESS Secondary Folding Tertiary Packing Quaternary Interaction  Protein Interaction occurs in the cytosol, in close proximity to other folded and packed proteins. involves interaction among tertiary structure elements of separate polymer chains Quaternary Structure non-linear 3 dimensional Many proteins are formed from more than one polypeptide chain. The quaternary structure describes the way in which the different subunits are packed together to form the overall structure of the protein. 3D structure of proteins Tertiary structure Quaternary structure  Class/Motif class = secondary structure composition, e.g. all , all , + motif = small, specific combinations of secondary structure elements, / e.g. -- loop both subset of fold  Fold fold = architecture = the overall shape and orientation of the secondary structures, ignoring connectivity between the structures, e.g. / barrel subset of fold families/superfamilies  Fold families/Superfamilies fold families = categorization that takes into account topology and previous subsets as well as biological properties, e.g. CLASS: + flavodoxin FOLD: sandwich FOLD FAMILY: flavodoxin superfamilies = in addition to fold families, includes evolutionary/ancestral  Hierarchical nature of protein structure Primary structure (Amino acid sequence) ↓ Secondary structure （ α-helix, β-sheet ） ↓ Tertiary structure （ Three-dimensional structure formed by assembly of secondary structures ） ↓ Quaternary structure （ Structure formed by more than one polypeptide chains ）  Watch this https://www.youtube.com/watch?v=qBRFIMcxZNM