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Protein Engineering. BIT 230. Protein Engineering. Design and construction of proteins by recombinant DNA techniques Michael Smith developed using oligonucleotide (site-directed) mutagenesis. Mutagenesis:Why Mutate?. Native proteins are not well suited for industrial application

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Protein engineering2 l.jpg
Protein Engineering

Design and construction of proteins by recombinant DNA techniques

Michael Smith developed using oligonucleotide (site-directed) mutagenesis


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Mutagenesis:Why Mutate?

  • Native proteins are not well suited for industrial application

  • Native proteins are not optimized for medicinal purposes

    • Increase the efficiency of enzyme-catalyzed reactions

    • Eliminate the need for cofactor in enzymatic reaction

    • Change substrate binding site to increase specificity

    • Change the thermal tolerance

    • Change the pH stability

    • Increase proteins resistance to proteases (purification)

    • Signal sequences - secretion

    • rare codon changes


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Aspargine Changes

If asparagine and glutamine present in protein

when heated, ammonia is released

amino acids convert to aspartic acid and glutamic acid

Protein may refold

LOSE ACTIVITY


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Adding Disulfide Bonds

Usually found in extracellular proteins, not intracellular

Cross link between chains or in chains formed by oxidation of cysteine residues

connective tissue

fibrin blood clots

Artificial addition may increase stability of protein

Avoid active site (enzyme)

Example:

xylanase

used to treat wood pulp in paper production

needs to function at high temp


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Reducing Free Sulfhydryl Residues

Cysteine residues may cause dimerization

through intermolecular disulfide bonding

Convert Cys to another amino acid (serine?)

reduce dimerization

maintain activity of enzyme


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Enzyme Activity and Specificity

  • Increase enzymatic activity by increasing affinity for enzyme

    • change sequences in substrate binding site

  • Change substrate of enzyme

  • tPA tissue plasminogen activator

    • dissolves blood clots



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