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Protein Engineering
BIT 230
Protein Engineering
Design and construction of proteins by recombinant DNA
techniques
Michael Smith developed using oligonucleotide (sitedirected) mutagenesis
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
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
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
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
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
Test, Test, Test