1. Protein Engineering Protein engineering Industrial enzymes (Table 8.1)
Generation of proteins suitable for industrial applications
Thermostability, resistance to organic solvent etc.
Industrial enzymes (Table 8.1)

2. Protein Engineering Adding disulfide bonds
Changing Asn to other amino acids
Reducing the number of free sulfhydryl residues
Increasing enzyme activity
Modifying metal cofactor requirements
Decreasing protease sensitivity
Modifying protein specificity
Increasing enzyme stability and specificity
Altering multiple properties

3. Adding Disulfide Bonds
Introduction of disulfide bonds
Increase in thermostability
In general, thermostability correlates with stability to organic solvent and other nonphysiological conditions
Selection of mutation sites
Disulfide bond formation between nearby residues
Choose mutation sites outside of the active site

4. T4 Ligase No. of S-S↑, Tm↑ S-S between 21 and 142
                          Amino acid at position:          
                        3     9     21    54    97    142   164   % Activity  Tm (oC)
   Wild type           Ile    Ile   Thr   Cys   Cys   Thr   Leu       100      41
   Pseudo wild type    Ile    Ile   Thr   Thr   Ala   Thr   Leu       100      41
   Variant A           Cys    Ile   Thr   Thr   Cys   Thr   Leu        99      47
   Variant B           Ile    Cys   Thr   Thr   Ala   Thr   Cys       101      48
   Variant C           Ile    Ile   Cys   Thr   Ala   Cys   Leu        0      49
   Variant D           Cys    Cys   Thr   Thr   Cys   Thr   Cys        98      58
   Variant E           Ile    Cys   Cys   Thr   Ala   Cys   Cys         0      58
   Variant F           Cys    Cys   Cys   Thr   Cys   Cys   Cys         0      66
No. of S-S↑, Tm↑
S-S between 21 and 142
 distortion of peptide backbone
 loss of enzyme activity

5. Changing Asn to Other Amino Acids
Asn and Gln at high temperature
Asn  Asp + NH3
Gln  Glu + NH3
Deamination becoming Asp and Glu
Localized change in peptide folding
 loss of activity
S. cerevisiae triosephosphate isomerase (Table 8.3)
Two Asn residues in subunit interface
Asn to Thr or Ile mutation  increase in thermostability and protease resistance
Asn to Asp mutation  decrease in stability
Long lasting human insulin
Mutation of an Asp to Gly

6. Reducing the Number of Free Sulfhydryl (-SH) Residues
Human INF-b
IFN-β produced in E. coli exists as dimmers and higher oligomers.
 activity↓
(Inactivation by intermolecular
disulfide bond formation)
Deduction of Cys residues involved in internal disulfide bond formation by comparison with IFN-a
Mutation of Cys 17 to Ser to prevent multimerization
Cys-17  Ser-17, activity↑

7. Increasing Enzyme Activity
Change of one or more amino acid residues of active site  activity↑
Change of Thr-51 to Ala-51 or Pro-51
(Table 8.4)
kcat/Km↑
kcat: catalytic rate constant
Km: binding constant (Km↓, binding affinity↑)

8. Modifying Metal Cofactor Requirements
Subtilisin
Protease produced by Bacillus
Used as cleaning agents in laundry detergent
Ca2+ binding is necessary for enzyme activity
Engineering of Ca2+-independent enzyme
Deletion of metal binding site
Selection potential stabilizing sites (possibly the sites previously interacted with the Ca2+-binding loop in the native form)
Random mutagenesis and screening for stable mutants
Combination of mutations

9. Decreasing Protease Sensitivity
Protease (plasmin) sensitivity of streptokinase
Mutation of cleavage sites
Lys 58, Lys 386  Gln (Gln has similar length of side chain to Lys)
21-fold increase in protease resistant

10. Modifying Protein Specificity
Restriction endonuclease
>2,500 enzymes but ~200 recognition sites
Rare cutters are necessary for producing large DNA fragments
Protein engineering of FokI endonulcease with zinc finger
FokI : nonspecific nuclease
Zinc finger:
Each zinc finger interacts with a specific DNA triplet codon.
Using a combination of zinc fingers, they will bind to a pre-determined site on a DNA fragment.
Expression as a fusion protein with zinc finger domains
To create unique endonucleases

11. Modifying Protein Specificity
Zinc Fingers
Expression as a fusion protein with zinc finger domains

12. Antibody Engineering Fab fragment Binding to antigenic determinant
FR (Framework Region)
CDR (Complementarity-Determining Region)
Hypervariable
Random mutagenesis with degenerate (mixed) primers and screening for new binding specificity

13. Random Mutagenesis of Three CDRs

14. Increasing Enzyme Stability and Specificity
Tissue plasminogen activator (tPA)
Serine protease
Dissolution of blood clots
Protein engineering of tPA
Increase in stability
Thr 103 to Asn
Increase in fibrin binding
Lys His Arg Arg ( ) to Ala Ala Ala Ala

15. Altering Multiple Properties
Subtilisin
DNA shuffling library from 26 different subtilisin genes
Screening of 654 clones in 96-well for desirable traits
Activity at 23oC
Thermostability
Solvent stability
pH dependence

16. Altering Multiple Properties
Peroxidase
From ink cap mushroom Coprinus cinereus
Dye transfer inhibitor in laundry detergent
Oxidizing (decolorizing) free dyes
Wash conditions using bleach-containing detergents
High pH, high temperature, high peroxide concentration
Protein engineering
Site-directed mutagenesis and random mutagenesis
DNA shuffling of mutants