1. Directed Mutagenesis and Protein Engineering

2. Improving Properties of Proteins for Therapeutic and Industrial Applications
Using a set of techniques that specifically change amino acids encoded by a cloned gene, proteins with properties that are better suited than naturally-occurring counterparts can be created for therapeutic and industrial applications.

3. Improving Properties of Proteins for Therapeutic and Industrial Applications
Increasing catalytic activity
Increasing stability at high temperature, extreme pH, or in organic solvent
Elimination of cofactor requirement
Increasing substrate specificity
Increasing resistance to cellular protease
Diminishing feedback inhibition by altering the allosteric regulation

4. Directed Mutagenesis ~ a process for generating amino acid coding
changes at the DNA level
Deciding the sites for mutagenesis
Based on structural information
Computer-assisted design
Site-directed mutagenesis using oligonucleotide
With M13 DNA
Using PCR
Random mutagenesis
With oligonucleotide analogues
Error-prone PCR
DNA shuffling

5. Oligonucleotide-Directed Mutagenesis with M13 DNA
Goal : ATTGGC  CTTGGC
Insert the cloned gene into the ds
M13 vector
Isolate the ss recombinant M13
vector
Add a synthetic oligonucleotide
containing a mismatched nucleotide
DNA polymerization
(Klenow fragment, dNTPs)
Ligation (T4 DNA ligase)
The ds DNA molecules are introduced
into E. coli (transformation)
Produce M13 plaques
Wild-type : mutated form = 1:1
<theoretically>

6. Mutagenesis with M13 DNA Recovery of the mutated DNA Expected: 50%
Result: 1~5% (for technical reasons)

7. Enrichment of mutated M13
Using a mutant E. coli (dut and ung)
dut mutation (defective dUTPase): dUTP ↑
 incorporation of dUTP (ⓤ) instead of dTTP
ung mutation (defective uracil N-glycosylase) : prevents the
removal of ⓤ ↓
In vitro mutagenesis
Transform wild-type E. coli ↘ The ung product removes ⓤ.
↘ A significant portion of the parental
strand is degraded. But the mutated
strand remains intact.

8. Enrichment of mutated M13

9. Oligonucleotide-Directed Mutagenesis Using Plasmid DNA

10. PCR-Amplified Oligonucleotide-Directed Mutagenesis

11. Random Mutagenesis with Degenerate Primers
To generate all the possible amino acid changes at one particular site
Using heterogeneous oligonucleotide primers synthesized with any of the four nucleotides at defined positions (Figure 8.5)
Random mutagenesis with degenerate oligonucleotide primers (Figure 8.6)

12. Chemical Synthesis of Heterogeneous Oligonucleotide Primers

13. Random Mutagenesis with Degenerate Primers

14. Random Mutagenesis with Nucleotide Analogues
ExoIII digests only the strand with the 3’ recessed end.
A mutated gene is created during replication in E. coli.

15. Error-Prone PCR
Ex) using Taq DNA polymerase, which lacks proofreading activity
The error rate may be increased by adding Mn2+ and unequal amounts of dNTPs
Error-prone PCR has been used to create enzymes with improved solvent and temperature stability and with enhanced specific activity

16. DNA Shuffling
Digestion of two or more of the native DNAs encoding similar proteins ↓
Ligation of the DNA fragments
(A large number of hybrids are generated.)

17. DNA Shuffling
Fragmentation of DNA (several members of a gene family) with DNase I (endonuclease) ↓
Selection of smaller DNA fragments
PCR without primers (DNA fragments cross-prime each other.)
PCR with ‘terminal primers’ to establish full-length hybrid DNAs.

18. PCR with “terminal primers” to obtain full-length DNAs (Fig. 8.11)
DNA Shuffling
Homology 1
Homology 3
Homology 2
PCR with “terminal primers” to obtain full-length DNAs (Fig. 8.11)
Homologous Genes
Fragmented Genes
Chimeric genes
DNase I digestion
Self-priming polymerase reaction

19. DNA Shuffling

20. Random mutagenesis, Error-prone PCR & DNA Shuffling

21. Mutant Proteins with Unusual Amino Acids
This E. coli contains the modified M. jannaschii Tyr-tRNA synthetase, which charges O-methyl-L-tyrosine-tRNA to amber suppressor tRNA