Protein Engineering Introduction & Applications

1. Protein
Engineering
Rekha
Ravindran

2. • Protein engineering is the design of new
enzymes or proteins with new or desirable
functions

3. • students must have a
clear understanding
of amino acid
chemistry and protein
structure (primary
and higher order), as
well as an
appreciation of the
range of stabilizing
forces that drives and
maintains protein
conformation nknown Author is licsed under CC BY-NC-ND

4. Pre-requisites
• Knowledge of Amino Acids
(Structure/Code/properties)
• Protein Structure Knowledge
(primary,sec,ter)
• Protein Synthesis inside cell (regulatory
elements)
• PTM
• Techniques (Structure/Sequencing)
• Software/Online tools

5. Case Studies • Basic enzymology
• protein glycosylation
• protein trafficking.
• basic knowledge of selected
analytical techniques,
specifically SDS-PAGE,
protein sequencing, and
isoelectric focusing.

6. Oxidation resistant
proteases
• A range of enzymes are
now added to clothing
deter- gents to help
remove biological-based
dirt such as food and
blood.

7. • Enzymes incorporated into detergents must
exhibit satisfactory stability and catalytic
activity in the presence of additional
detergent components (including surfactants
and oxidizing agents) and washing conditions
(e.g. high pH as well as temperatures
approaching or surpassing 60 °C).

8. A subset of serine proteases, the subtilisins

9. catalytic triad composed of serine, histidine, and
aspartate residues, usually at/near positions
221, 64, and 32, respectively.
This Photo by Unknown Author is licensed under CC BY-SA

10. • A methionine residue is invariably found immediately
(usually at position 222).
• Surface methionine are susceptible to oxidation by
detergent bleach
• Oxidation usually severely reduces catalytic activity.

11. • The effect of an oxidizing agent (H2O2) upon
the activity of a native subtilisin and an
engineered subtilisin rendered oxidation-
resistant by replacement of Met222 with ?

12. CASE STUDY 2: THE ENGINEERING OF TPA
• Tissue plasminogen activator (tPA)1 is a 527-
amino acid, 70-kDa serine protease
• found application as a thrombolytic agent in
the treatment of heart attacks and strokes.
This Photo by Unknown Author is licensed under CC BY-SA

13. • Retavase, is an engineered, domain-deleted
variant of native tPA, consisting only of the
catalytic (P) and kringle 2 (K2) domains. The
engineered nucleotide construct is expressed
in Escherichia coli; thus the product is also
unglycosylated

14. • The P and K2 domains provide the engineered
molecule with catalytic activity against fibrin
(which forms the structural basis of a blood
clot). The absence of the epidermal growth
factor and K1 domains, along with the
glycocomponent, confers a significantly
extended plasma half-life upon the molecule
(19 min), facilitating its convenient clinical
administration via a single intravenous
injection.

15. • The engineered tPA product is unglycosylated.
If you wished to produce a glycosylated
product variant, how might you do so?

16. ENGINEERED INSULINS
• Novorapid and Novolog are trade names of
engineered product in which the proline at
position B 28 has been replaced with an
aspartate residue, thereby introducing charge
repulsion at the monomer-monomer surface
This Photo by Unknown Author is licensed under CC BY

17. • Sequence structure function

18. Applications
• Food Industry
• Detergent industry
• Environmental applications
• Medical Applications
• Virus engineering

19. Applications
• Design new enzymes- Thermostability and
catalytic efficiency
• Food processing enzymes such as amylases
and lipases, the properties of which are
improved using recombinant DNA technology
• Modify antibodies to target cancer cells for
clinical applications
• Producing new biomaterials for medical and
engineering applications.

20. • In 1978, a very small amount of Ricin was used
by the KGB to kill a Bulgarian Soviet political
dissident in London.Many organisms produce
toxic proteins that exert lethal activity towards
other cells. Ricin is an extremely potent
protein toxin produced by the castor bean
plant and has no known antidote. A single
molecule will inactivate almost 2000
ribosomes within a minute.
s Photo by Unknown Author is licensed under CC BY-SA

21. • The potency of Ricin has found beneficial
application in the field of chemotherapy as a
component of ‘immunotoxin’ conjugates, but
the toxin also has been exploited as a poison
for biological warfare and bioterrorism.
• https://academic.oup.com/peds/article/17/4/
391/1672228
This Photo by Unknown Author is licensed
under CC BY

22. • RiVax is a recombinant protein that is
currently under clinical development as part
of a human vaccine to protect against ricin
poisoning.

24. This Photo by Unknown Author is licensed under CC BY-SA

32. Amino acids

33. • Charged Residues are the most polar of all. They are
seldom buried in the interior of a folded protein. They are
normally found on the surface of the protein where they
interact with water and with other important biological
molecules. these groups can be important in the
recognition (binding) of oppositely charged groups on
molecules that interact with proteins.
• Polar Residues are both buried as well as on the
surface of the protein. They either form hydrogen bonds
with other polar residues in the protein or with water on
the surface. For example, the OH group of serine can both
donate as well as accept a hydrogen bond:

34. • Hydrophobic groups. some of the R groups are
hydrophobic, seeking to avoid contact with water at
all costs. These amino acids are often buried in the
protein interior where they play a key role in
stabilizing the protein’s three-dimensional structure.
This leads to reduced mobility within the protein
interior and holds together the protein structure

36. • Secondary Structure
• Secondary structure is the ordered
arrangement or conformation of amino acids
in localized regions of a polypeptide or protein
molecule. Hydrogen bonding plays an
important role in stabilizing these folding
patterns. The two main secondary structures
are the alpha helix and the anti-parallel beta-
pleated sheet

38. • Some tertiary protein structures are
constituted of a single domain, others contain
several domains with similar or different folds.
Protein domains are the basic classification unit
of protein folds.

39. • The tertiary structure of proteins is the result of further bonding between
side chains within the protein and with any water that may be present around
the protein. Polar amino acids move to the outside of the shape and non-
polar amino acids move to the inside when placed in a polar solution. Bonds
that are considered part of the tertiary structure include:
1. Bonds formed between non-polar side chains,
2. Disulfide bonds formed between sulfur atoms in cysteine side chains,
3. Ionic bonds formed between acidic and basic side chains, and
4. Hydrogen bonds formed between carbonyl groups and hydroxyl or amino
groups