protein engineering and site directed mutagenesis

1. Protein Engineering and site
directed mutagenesis
Dr. Nawfal Hussein
Email: nawfal_hm@yahoo.com
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2. Protein Engineering
 Protein Engineering: The sequence of the protein may be
altered by genetic engineering to improve its properties.
 proteins are used under relatively harsh conditions and are
exposed to oxidizing conditions not found inside living cells.
Consequently, these particular proteins are unusually robust
and stable and are not at all representative of typical enzymes in
this respect.
 Introduction of extra disulfide bonds, by altering the coding
sequence for an enzyme, often provides major increases in
protein stability.
 A variety of alterations to the amino acid sequence may yield
proteins with moderate increases in stability.
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3.  Altering amino acids in and around the active site may change
the specificity of an enzyme toward substrates or cofactors.
 Many proteins are larger than necessary from the industrial
viewpoint. Removing extra sequences and simplifying the
protein may increase industrial efficiency.
 Proteins may be altered by directed evolution, which consists of
random mutagenesis of the coding sequence, followed by
biological selection for improved or novel properties.
 Many proteins consist of discrete domains with individual
functions. Recombining domains from different proteins can
generate hybrid proteins with novel combinations of properties.
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4.  In DNA shuffling, the coding sequence for a protein is
rearranged in the hope of generating novel or improved
activities. Mutations may also be introduced during the
procedure to provide more variation.
 Another shuffling technique to generate novel proteins is to use
premade protein modules. The coding sequences for the
modules are combined in various combinations by a PCR-based
approach.
 Semirandom DNA sequences may be used to generate libraries
of de novo proteins. These may then be screened for possible
binding activities or biological functions. Several de novo proteins
have been found that can replace natural proteins.
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5.  A variety of schemes have been devised to insert non-naturally
occurring amino acids into proteins. The most sophisticated
schemes consist of changing the coding properties of selected
codons.
 Non-natural amino acids may be used for a variety of
experiments. Some provide reactive groups for attachment or
cross-linking, whereas others act as spectroscopic probes.
 Biomaterials for medical use, especially extracellular proteins
used in reconstructive surgery, may be improved by protein
engineering.
 Creating binding proteins for a variety of uses is still in the
experimental stages. However, it may eventually prove possible
to replace cumbersome antibodies with smaller and more stable
proteins.5

6. in vitro mutagenesis
 Technique that enables a directed mutation to be made at a
specific point in a cloned gene.
 Is protein structure affects the action of an enzyme?
 identity of the amino acids that provide the substrate binding
and catalytic functions of an enzyme molecule
 Role of each individual amino acid to be assessed by replacing
it with an alternative residue and determining the effect this has
on the enzymatic activity
 Alterations to the amino acid sequence of subtilisin, an enzyme
used in biological washing powders, have resulted in
engineered versions with greater resistances to the thermal
and bleaching (oxidative) stresses encountered in washing
machines.
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7. Types of in vitro mutagenesis techniques
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 A restriction fragment can be deleted

8.  The gene can be opened at a unique restriction site, a few
nucleotides removed with a double-strand-specific
endonuclease such as Bal31, and the gene religated
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9.  A short, double-stranded oligonucleotide can be inserted at a
restriction site. The sequence of the oligonucleotide can be such
that the additional stretch of amino acids inserted into the
protein produces, for example, a new structure such as an a-
helix, or destabilizes an existing structure.
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11. In vitro DNA synthesis
 Single-stranded DNA is sometimes generated for sequencing by using
M13 vectors. In vitro DNA synthesis may be performed using such
single-stranded DNA as template using T7 polymerase and a supply of
nucleoside triphosphates. DNA polymerization may be initiated using
artificially synthesized primers whose sequence has been altered by a
few bases. This will generate a mutagenized product that incorporates
these changes.
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12. PCR based mutagenesis
 a) Introduction of specific base changes
Using PCR primers whose sequence has been altered will generate a
PCR product that incorporates these changes.
 b) Localized random mutagenesis
Manganese ions cause errors in PCR reactions. Hence, random
mutations may be introduced into the segment of DNA being
amplified.
 c) Generation of insertion or deletion by PCR
Using PCR primers that include sequences homologous to the target
location allows replacement of a region of chromosome with a
segment of DNA generated by PCR.
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14. Transgenic technology
 Transgenic technology creates genetically-modified organisms. It may
therefore be regarded as a form of mutagenesis. Extra DNA
sequences may be introduced from other organisms by a variety of
techniques.
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