Protein Engineering

BIT 230

Protein EngineeringDesign 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

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 releasedamino acids convert to aspartic acid and glutamic acidProtein may refoldLOSE ACTIVITY

Adding Disulfide BondsUsually found in extracellular proteins, not intracellular

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

connective tissuefibrin blood clots

Artificial addition may increase stability of protein

Avoid active site (enzyme)

Example:xylanase

used to treat wood pulp in paper productionneeds to function at high temp

Reducing Free Sulfhydryl Residues

Cysteine residues may cause dimerizationthrough intermolecular disulfide bonding

Convert Cys to another amino acid (serine?)reduce dimerizationmaintain 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