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Protein structure

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But they differ in their state of assembly and functional capacity. ... ampicillin (amp), tetracyclin (tet) or Cloramphenicol (Cam) or Kanamycin (Kan) Cloning sites ... – PowerPoint PPT presentation

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Title: Protein structure


1
  • Protein structure
  • Polypeptide and proteins- Both described
    molecules composed of amino acids. But they
    differ in their state of assembly and functional
    capacity. As assembled on the ribosome during
    translation the molecule is named as polypeptide.
    After released from the ribosome the polypeptide
    folds up and assumes a higher order structure.
  • The two most common secondary structure
    arrangements are the right-handed a-helix and the
    b-sheet, which can be connected into a larger
    tertiary structure (or fold) which defines the 3
    dimensional conformation of the entire chain in
    space.
  • When the final conformation is achieved only the
    molecule becomes functional and is called a
    protein. It is the 3 dimensional conformation
    that is essential to the function of the protein

2
  • The quarternary level of organization applies
    only to proteins composed of more than one
    polypeptide chains. This type of protein is
    called oligomeric and each chain is called a
    subunit eg. Hemoglobin, DNA and RNA polymerase
    have quarternary structures.
  • Posttranslational modifications of proteins
  • The N-terminus and C terminus amino acids are
    usually removed or modified. Eg. N terminal
    formylmethionine of bacterial polypeptide is
    usually removed.
  • Individual amino acid residues are sometimes
    modified. Addition of methyl groups or phosphates
    to the hydroxyl group Kinases are responsible for
    addition of PO4.
  • Carbohydrate side chains are sometimes added,
    glycoproteins
  • Polypeptide chains are often complexed with metal
    ions.

3
  • Electrophoresis of nucleic acids
  • Electrophoresis is a technique used to separate
    macromolecules - especially proteins and nucleic
    acids - that differ in size, charge or
    conformation. As such, it is one of the most
    widely-used techniques in biochemistry and
    molecular biology.
  • When charged molecules are placed in an electric
    field, they migrate toward either the positive
    (anode) or negative (cathode) pole according to
    their charge. In contrast to proteins, which can
    have either a net positive or net negative
    charge, nucleic acids have a consistent negative
    charge imparted by their phosphate backbone, and
    migrate toward the anode.
  • Proteins and nucleic acids are electrophoresed
    within a matrix or "gel, agarose or
    polyacrylamide
  • Ethidium bromide, a fluorescent dye is used for
    staining nucleic acids and DNA bands are visible
    under UV light.

4
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5
  • Recombinant DNA Technology
  • DEFINITION OF RECOMBINANT DNA
  • DNA molecules constructed outside of living cells
    by joining natural or synthetic DNA segments to
    DNA molecules that can replicate in a living cell
  • Restriction endonuclease Any one of several
    enzymes, produced by bacteria, that break foreign
    DNA molecules at very specific sites. Extensively
    used in recombinant DNA technology. Restriction
    Enzymes are made to protect the bacteria from
    foreign DNA. Bacteria have a method of marking
    their own DNA as being "self" (called a
    Modification System). Any DNA not recognized as
    self is digested into smaller pieces by the
    Restriction Enzymes
  • Recognition site The specific site at which
    restriction endonucleases cleave DNA is defined
    by a sequence of bases.

6
  • Restriction Enzymes search for exact sequences of
    a defined length (recognition sites). Some
    enzymes recognize sequences 4 bp long (e.g.,
    GTAC), some 6 (e.g., GAATTC), and still others 8
    or more.

7
  • One of the common features of most enzyme
    recognition sites is that they are palindromes. A
    palindrome is a sequence which is read the same
    on both strands in the 5' --gt 3' direction.
  • For Res. Enzyme EcoRI 5 GAATTC 3
  • 3
    CTTAAG 5

  • This type of cut is called staggered, because it
    results in fragments with single-stranded ends.
    The single-stranded ends are said to be sticky
    because they are able to bind to a complementary
    single-stranded region
  • According to the pattern of cleavage Res. Enzymes
    results in 3 types of DNA fragments
  • 5 overhangs 5 G.----------3
  • 3 CTTAAG 5

8
  • 3 overhangs 5 GAATTC 3
  • 3 C---------- 5 these
    make sticky ends
  • Blunt ends 5 GTT.3
  • 3 CAA..5
  • Different Res. Enzymes recognize and cleave
    different of bases
  • Eg. 4 base cutters, 5,6,7 etc ex. EcoRI is a 6
    base cutter
  • How often will a RE cut a random DNA fragment?
  • A 4 base cutter 1/4x1/4x1/4x1/4 1/256 bp of DNA
    (one in every 256 base pairs of DNA)
  • The general rule is (1/4)n where n is the of
    nucleotide bases in the restriction site.
  • These sticky ends can reanneal with complementary
    single stranded tails on other DNA fragmets. If
    mixed under the proper conditions, DNA fragments
    from two sources form recombinant molecules and
    DNA ligase links the two fragments.

9
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10
  • DNA cloning and cloning vectors
  • What is molecular cloning? Genetically
    engineered replicates of DNA sequences
  • Why clone DNA ?
  • To identify an individual gene
  • To isolate a gene
  • To clone genomic DNA
  • To make clones made from mRNA, called cDNA clones
  • To construct genomic libraries/cDNA libraries
  • Genomic libraries are collections of clones that
    may contain at least one copy of every DNA
    sequence from the genome of interest.
  • Cloning vectors Vehicles that enable the
    replication of cloned fragments. Most have some
    general features in common.
  • - small and well characterized genomes that
    contain an origin of replication to enable the
    molecule to replicate itself and the insert
    fragment.

11
  • All cloning vectors must have 4 common features
  • An origin of repliation (Ori site)
  • A dominant selectable marker, usually for
    antbiotic resistance I.e.
  • Unique restriction enzyme sites should be
    available
  • Must be easy to recover from a bacterial host
  • Plasmids- These are molecules of DNA that are
    found in bacteria separate from the bacterial
    chromosome. They
  • are small (a few thousand base pairs)
  • usually carry only one or a few genes
  • are circular ds DNA molecules,
  • have a single origin of replication
  • In nature, these plasmids often encode genes for
    proteins (e.g., enzymes) that protect the
    bacterium from one or more antibiotics.

12
  • Plasmids were The first vectors to be genetically
    modified and used in cloning
  • To use them as vectors they should be modified
    to contian a limited number of res. Sites and
    selectable marker genes that detect the presence
    of plasmid in the host cell.
  • Selectable markers
  • When you transform bacteria, not all cells will
    receive copies of the plasmid
  • Selectable marker genes allow for easy
    identification of transformed cells
  • The most common selectable markers are antibiotic
    resistance genes. ampicillin (amp), tetracyclin
    (tet) or Cloramphenicol (Cam) or Kanamycin (Kan)

13
  • Cloning sites
  • These are regions of DNA which contain the
    consensus sequence for numerous restriction
    enzymes.
  • Called the multiple cloning site (MCS) or
    polylinker
  • The restriction enzymes that cut within the MCS
    do not cut elsewhere in the vector
  • This allows for target DNA to be inserted into a
    specific part of the vector.
  • Screening of recombinants- once the DNA of
    interest is inserted to the plasmid at the
    restriction site in the polylinker the
    recombinant plasmid then have to be transformed
    into bacteria. Within the bacteria these plasmids
    produce large number of copies through its
    replication process.
  • Bacteria cells are plated on media containig the
    appropriate antibiotic, IPTG (a lactose analog)
    and X-gal. When induced by IPTG the galactoside
    gene will be transcribed and the enzyme will

14
  • Use X-gal as a substrate.
  • When X-gal is cleaved it produces a blue pigment.
    When there is an insert in the polylinker lacZ
    transcription is disturbed no B galatosidase is
    formed and the X-gal not cleaved.
  • Colonies with inserts will be white

15
  • One such plasmid is pUC18, which is small (2686
    bp) and allows to carry relatively large DNA
    inserts. In a host cell it replicates 500 copies
    per cell, producing many copies of inserted DNA
    fragments.
  • A large of restriction sites are available in a
    polylinker site.
  • It has a selection system to identify the
    recombinant plasmids.
  • The pUC 18 carries the bacterial lacZ gene. When
    grown in a medium of X-gal it produces a blue
    colony. The polylinker is inserted within the
    lacZ gene. When a DNA fragment is inserted into
    the polylinker site, the lacZ gene is
    inactivated. Therefore the recombinant plasmids
    when grown in X-gal would produce white colonies.
  • One disadvantage in plasmid vectors is it can
    carry up to 10 kb of inserted DNA. For
    experiments when larger DNA fragments are
    required genetically modified strains of lambda
    phage are used as vectors.

16
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17
  • Cloning in E.coli host cells- one of the most
    commonly used hosts is a laboratry strain of
    E.coli known as K12.
  • Lambda phage vectors are ds virusDNA that
    infect E.coli cells. These bacteriophage ?
    cloning vectors has a chromosome with a left and
    right arm that collectively contain all the
    essential genes for the lytic cycle. Between the
    two arms is a dispensable segment of DNA with
    EcoRI sites. This dispensable region is replaced
    by the DNA insert. They are then inserted into
    bacterial host cells where they reproduce to form
    many infected particles of phages, each carrying
    a DNA insert. Phage vectors can carry about 20 kb
    DNA inserts, and useful when cloning entire
    genomes. Fig 16.9

18
  • . Cosmids Cosmids are vectors which have
    characteristics of both viruses and plasmids.
    They contain the phage cos site necessary for
    packaging DNA into phage heads. Most of the
    lambda genes have been deleted.
  • 1) Can accept 40-50 kb inserts.
  • 2) Recombinant cosmids are packaged into phage
    heads and the particles used to infect cells.
  • 3) Once inside a cell, the DNA replicates as a
    plasmid
  • . Shuttle Vectors
  • 1) Designed to replicate and function in two
    different kinds of cells
  • 2) E. coli and yeast
  • 3) E. coli and Agrobacterium
  • Cloning in Eukaryotic host cells The yeast
    Saccharomyces cerevisiae is extensively used as a
    host cell for growth and expression of cloned
    eukaryotic genes.

19
  • Yeast Artificial chromosomes (YAC)
  • Eukaryotic chromosomes have common features that
    enable them to successfully replicate in a cell.
    They have
  • Two telomeres
  • A centromere
  • An origin of replication along the chromosomes
    (ARS)
  • YAC are cloning vectors that have been
    constructed so that you can add inserts between
    the telomeric regions and effectively clone very
    large regions of DNA.
  • YAC vectors have A selectable marker at each
    end TRPI (tryptophan independence) and URA3
    (Uracil independence) and a restriction enzyme
    site.
  • Using the above procedures, many human genes have
    been cloned in E. coli or in yeast. This has made
    it possible - for the first time - to produce
    unlimited amounts of human proteins in vitro.
    Cultured cells (E. coli, yeast, mammalian cells)
    transformed with the human gene are being used to
    manufacture

20
  • Insulin for diabetics
  • Factor VIII for males suffering from hemophilia A
  • Factor IX for hemophilia B
  • Human growth hormone (GH)
  • erythropoietin (EPO) for treating anemia
  • three types of interferons
  • several interleukins
  • adenosine deaminase (ADA) for treating some forms
    of severe combined immunodeficiency (SCID)
  • Parathyroid hormone and many more
  • Cloning without host cells
  • Polymerase Chain Reaction (PCR) Developed in
    1986 this is a rapid method of DNA cloning and
    very much used instead of vectors

21
  • Polymerase chain reaction is a technique that
    amplifies DNA, which enables to make millions -
    or even billions - of copies of a DNA molecule in
    a very short time.
  • PCR has been the most widely used technique in
    the field of molecular biology since it was
    developed in 1986.
  • It has been used to detect DNA sequences, to
    diagnose genetic diseases, to carry out DNA
    fingerprinting, to detect bacteria or viruses
    (particularly the AIDS virus), and to research on
    human evolution. It has even been used to clone
    the DNA of an Egyptian mummy!
  • PCR Methodology
  • In order to perform PCR, you must know at least a
    portion of the sequence of the DNA molecule that
    you wish to replicate
  • You must then synthesize primers short
    oligonucleotides (containing about two dozen
    nucleotides) that are precisely complementary to
    the sequence at the 3' end of each strand of the
    target DNA (DNA you wish to amplify ) (Fig.
    16-11)

22
  • The DNA sample is heated to separate its strands
    denaturation (denature at 90-95c) and mixed with
    the primers-.
  • Temp. is then lowered to 50-70c when primers
    would anneal, if they find their complementary
    sequences in the flanking regions of the target
    DNA- annealing.
  • Synthesis begins (as always 5' -gt 3') using the
    original strand as the template(Temp. between
    70-75c).
  • The reaction mixture must contain all four
    deoxynucleotide triphosphates (dATP, dCTP, dGTP,
    dTTP), DNA polymerase and Mg2. A special kind
    of DNA polymerase is used that is not denatured
    by the high temperature needed to separate the
    DNA strands, the enzyme Taq polymerase is
    obtained from the heat stable bacteria Thermus
    aquaticus
  • Taq polymerase extends the primers and
    polymerization continues until each
    newly-synthesized strand has proceeded far enough
    to contain the site recognized by the other
    primer- extension.

23
  • In this way two DNA molecules identical to the
    original molecule is formed and the cycle is
    repeated.
  • Each cycle doubles the number of DNA molecules.
  • Using automated equipment (Thermocycler), each
    cycle of replication can be completed in less
    than 5 minutes. After 30 cycles, what began as a
    single molecule of DNA has been amplified into
    more than a billion copies (230 1.02 x 109).
  • Advantages of PCR
  • Requires very minute amounts of DNA
  • Little DNA purification is needed.
  • Partially degraded DNA can be used
  • Extremely rapid diagnostic potential and very
    sensitive
  • Amenable to many analytical procedures
  • Can be largely automated

24
  • Limitations in PCR
  • Generated sequences may have errors
  • False results possible if DNA is contaminated
  • Some applications have reproducibility problems

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