directed mutagenesis and protein engineering

1: Directed Mutagenesis and Protein Engineering Sometimes the naturally occurring protein simply is not well suited for industrial purposes Modifications previously done by random mutagenesis (tedious, limited success at times) Now done by directed mutagenesis

2: Examples of Properties Altered for Improved Therapeutic or Industrial Applications Michaelis constant (Km), Vmax, catalytic rate constant (kcat) Thermal tolerance, pH stability Reactivity in nonaqueous solvents Eliminate cofactor requirement Alter substrate binding site/specificity Increase protease resistance Alter allosteric regulation

3: Oligonucleotide-directed Mutagenesis Synthesize oligonucleotide with desired nucleotide change Bind to ssDNA template Replicate DNA Transform Isolate plaques with mutant sequence Low success rate due to cellular correction of mismatch

4: Enrichment for Mutant M13 Clones Make ssDNA vector in dut ung mutant strain Uracil substituted for thymine Mutagenesis as before with “new strand” using thymine Transform Cell corrects mismatch while degrading uracil contain strand

5: Directed Mutagenesis Using Plasmids Coselection technique Antibiotic resistance gene restored and a second one eliminated Approx 90% of Ampr and Tets clones have target mutated in directed fashion

7: Random Mutagenesis Using Degenerate Oligonucleotide Primers Make primers at sites to be mutated which are degenerate at one or more sites

8: Random Mutagenesis Using Degenerate Oligonucleotide Primers

9: Random Mutagenesis With Nucleotide Analogs Cut clone to side of target gene Digest with ExoIII Synthesize with DNAP I, dNTPs and a dNTP analog Analog causes replication errors when transformed into E. coli

10: Mutagenesis by Error-Prone PCR Taq DNAP I lacks proofreading activity Manganese ions increase error rate Transform with PCR products Screen for desired phenotype

12: DNA Shuffling Uses families of related genes Assemble “new” genes from combinations of restriction fragments of naturally occurring genes Method used to make improved interferons

13: PCR-Based DNA Shuffling Mix sets of cut fragments Cross priming occurs Terminal primers then give full length fragments Much quicker than ligating restriction fragments

14: Insertion of Novel Amino Acid Analogues Alter targeted codon to TAG (UAG) Transform into strain with modified tRNA and aminoacyl tRNA synthetase

15: Industrial Use of Enzymes Although thousands of enzymes are used for industrial purposes, only about 20 make up 90% of the use Modifying these enzymes can yield improvements in the heavily utilized processes

16: Industrial Enzymes and Their Commercial Uses

17: Adding Disulfide Bonds Increases thermostability Can improve organic solvent resistance and pH stability Decreased flexibility can reduce activity, too Sometimes possible changes are done in a “trial and error” fashion and then evaluated individually

18: Properties of T4 Lysozyme and Six Engineered Variants

19: Engineered Disulfide Linkages Xylanase Degrades hemicellulose, reducing the need for bleaching wood pulp fiber Need for temperature stable variant Computer modeling suggested sites for SS bonds Improved variant produced Human pancreatic ribonuclease Antitumoragenic agent, dimeric structure To reduce antigenic problems human enzyme engineered to form dimer

20: Engineered Human Pancreatic RNase Cysteines added to allow for disulfide bond formation E. coli placed the protein product in inclusion bodies (not good)

21: Denaturation/Renaturation of RNase Pancreatic RNase is readily denatured/renatured Restored activity of product from inclusion bodies

22: Changing Asparagine When asparagine deaminates protein can lose activity Some other amino acids can be successfully substituted which maintain enzyme activity and are not deaminated

23: Stability at 100ºC of Yeast Triosephosphate Isomerase and Engineered Derivatives

24: Reducing Free Sulfhydryl Residues Free sulfhydryl groups allow dimer/oligomer formation, especially at high protein concentrations and upon storage Change cys to acceptable alternative amino acid (e.g. ser)

25: Increasing Enzyme Activity Modify catalytic function such as substrate specificity Tyrosyl-tRNA synthetase Structural data very detailed Amino acid change to alter ATP binding Catalytic efficiency improved

26: Aminoacylation Activity of Native (THR-51) and Modified (Ala-51 and Pro-51) Tyrosyl-tRNA Synthetases

27: Modifying Metal Requirements Serine protease for laundry detergent Require calcium Calcium induces conformation change necessary for activity Modify amino acid sequence to achieve conformation and stability without calcium

28: Effect of random mutations of selected amino acid residues on the stability of subtilisin BPN’ lacking a calcium binding domain

29: Decreasing Protease Sensitivity Streptokinase used to activate plasmin to break up blood clots Plasmin also destroys streptokinase Plasmin cuts after lys or arg Make streptokinase plasmin-resitant by eliminating unnecessary lys residues

30: Modifying Protein Specificity Nuclease domain of FokI restriction endonuclease Target to specific DNA sequence using sequence specific DNA binding zinc finger domains

31: Modifying Antibodies Complementarity-determining regions Directed mutagenesis of CDRs gives antibodies with new specificities Mutagenesis can be random (e.g. error-prone PCR or mutagenic primers) or highly specific based upon predicted protein structure

32: Introducing Changes into CDRs Multiple PCR amplifications using mutagenic primers

33: Screening Shuffled Libraries Shuffle 26 different subtilisin genes Transform chimeric molecules Plate on agar containing milk proteins Look for zones of clearing Assay under various conditions in 96 well plates Can be done with mutant variations of single gene, too (e.g. peroxidase)

34: Shuffling of Laboratory-Produced Mutant Variants

35: Improving Enzyme Stability and Specificity Tissue plasminogen activator (tPA) used to dissolve blood clots tPA rapidly cleared from bloodstream High tPA concentrations caused nonspecific bleeding Modify amino acids to increase half-life in bloodstream Modify to increase fibrin specificity and decrease side effects

36: Stability and Activity of Various Modified Versions of tPA