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CHEMICAL BIOLOGY
Modifikasi Target Makromolekul

Disampaikan oleh:
Dr. apt. Rumiyati, M.Si
 Protein Engineering
- Modifikasi protein -
• Mutagenesis used for modifying proteins

• Replacements on protein level -> mutations on DNA level

• Assumption : Natural sequence can be modified to

improve a certain function of protein

This implies:
• Protein is NOT at an optimum for that function
• Sequence changes without disruption of the structure
(otherwise it would not fold)
• New sequence is not TOO different from the native sequence
(otherwise loss in function of protein)
consequence -> introduce point mutations
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 Protein Engineering
to obtain a protein with improved or new properties

Rational Protein Design Nature

Proteins with Novel Properties

Random Mutagenesis

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 Rational Protein Design
 Site –directed mutagenesis !!!

Requirements:

-> Knowledge of sequence and preferable Structure

(active site)

-> Understanding of mechanism

(knowledge about structure – function relationship)

-> Identification of cofactors

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 Protein Engineering

What can be engineered in Proteins ?

-> Folding (+Structure):

a. Thermodynamic Stability
(Equilibrium between: Native  Unfolded state)

b. Thermal and Environmental Stability (Temperature, pH, Solvent,

Detergents, Salt …..)

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 Protein Engineering
What can be engineered in Proteins ?

-> Function:
1. Binding (Interaction of a protein with its surroundings)

How many points are required to bind a molecule with high affinity?

2. Catalysis (a different form of binding – binding the transition state

of a chemical reaction)

Increased binding to the transition state  increased catalytic rates !!!

Requires: Knowledge of the Catalytic Mechanism !!!

-> engineer Kcat and Km

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 Protein Engineering

a. Factors which contribute to stability:

1. Hydrophobicity (hydrophobic core)

2. Electrostatic Interactions:

-> Salt Bridges

-> Hydrogen Bonds
-> Dipole Interactions

3. Disulfide Bridges

4. Metal Binding (Metal chelating site)

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 Protein Engineering

b. Design of Thermal and Environmental stability:

1. Stabilization of -Helix Macrodipoles

2. Engineer Structural Motifes (like Helix N-Caps)

3. Introduction of salt bridges

4. Introduction of residues with higher intrinsic properties for their

conformational state (e.g. Ala replacement within a -Helix)

5. Introduction of disulfide bridges

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Protein Engineering - Applications

Engineering Stability of Enzymes – T4 lysozyme

-> S-S bonds introduction

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 Protein Engineering - Applications

Engineering Stability of Enzymes – triosephosphate isomerase from yeast

-> replace Asn (deaminated at high temperature)

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 Protein Engineering - Applications

Engineering Activity of Enzymes – tyrosyl-tRNA synthetase from B.

stearothermophilus

-> replace Thr 51 (improve affinity for ATP) -> Design

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Protein Engineering - Applications
Engineering Ca-independency of subtilisin

Saturation mutagenesis -> 7 out of

10 regions were found to give
increase of stability

Mutant:
10x more stable than native
enzyme in absence of Ca
50% more stable than native in
presence of Ca

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 New therapeutics from Recomb-DNA
More than 200 products
• Insulin
• Human growth hormon (hGH)
• Tissue type plasminogen activator (tPA)
• IL-2
• INF

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 Therapeutic proteins
• Insulin is part of a class of proteins called hormones
• It is produced by cells in our pancreas and secreted
into the bloodstream to stimulate uptake of blood
glucose into body cells such as muscle tissue
• Allowing blood glucose levels to remain high causes
health problems
• high blood pressure
• poor blood circulation
• cataracts

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 Insulin
(synthesis, storage, secretion)
• Produced within the pancreas by β
cells  islets of Langerhans
• insulin mRNA is translated as a single
chain precursor called preproinsulin
• removal of signal peptide during Zn
insertion into the endoplasmic
reticulum generates proinsulin
• Within the endoplasmic reticulum,
proinsulin is exposed to several
specific endopeptidases which excise
the C peptide, thereby generating the
mature form of insulin
• Stored as β granules
 Insulin
• Discovered in 1921 by Banting and Best
• Consist of A & B chains linked by 2 disulfide bonds
(plus additional disulfide in A)

 A = 21amino acids B = 30 amino acids

Insulin drug evolution
Stage 1 Insulin was extracted from the glands of cows
and pigs. (1920s)

Stage 2 Convert pig insulin into human insulin by

removing the one amino acid that distinguishes them and
replacing it with the human version.
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 Stage 3 Insert the human
insulin gene into E. coli and
culture the recombinant
E.coli to produce insulin
(trade name = Humulin®).
Yeast is also used to produce
i n s u l i n ( t r ad e n a me =
Novolin®) (1987).

Recombinant DNA technology has also made it possible to

manufacture slightly-modified forms of human insulin that work
faster (Humalog® and NovoLog®) or slower (Lantus®) than
regular human insulin.
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Types of insulin

• Regular insulins
• Insulin analogs
• Pre-mixed insulin
Short peptide mimics

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Regular insulins:

• Human insulin: Humulin® (from E.coli),

Novalin® (from yeast)
• NPH - neutral protamine Hagedorn (NPH),
protamine mixed.
• Lente® insulin / Ultralente® insullin-
zinc added

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Insulin Analogs:

• Fatty Acid Acylated insulins

• Insulin Lispro (Humalog®) (1996)

• Insulin Aspart (NovoLog®) (2000)

• Insulin Glargine (Lantus®) (2002)

• Insulin Detemir (Levemir®) (Jun.,2005)

• Insulin Glulisine (Apidra®) (Jan., 2006)

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 Amino Acid Substitutons

A- chain B- chain Position

Position

Source/ A21 B3 B28 B29 B30 B31

Type And
B32
Human Asn Asn Pro Lys Thr
Aspart Asn Aspartic Lys Thr
acid rapid-acting
Lispro Asn Lys Pro Thr
Glulisine Asn Lys Pro Glu Thr

Glargine Gly Pro Lys Thr Arg

long-acting
Detemir Lys Myristic
acid 23
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 Challenging..

• Protein pharmaceuticals is a rapidly expanding area.

• Large size and poor stability of proteins makes formulation
and delivery more challenging than with most conventional
drugs.
• The parenteral route is traditionally the most used route of
administration.
• Non-invasive delivery systems are emerging, but very few
are currently on the market.

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Interferon
• Clasification : IFN, IFN,  synthesized in cells that have been
exposed to viruses or viral RNA
• IFN   synthesized in response to cell growth stimulating agents
• IFN  IFN2 IFN1 similar effect in virus-challenging bovine cell line.
• IFN2 7x effective > IFN1 in human cell treated with virus

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• Hybrid genes were constructed in an effort to create
proteins with novel interferon activity

RE RE RE

IFN2

IFN3

RE RE RE

Hybrid
genes

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Human Growth Hormone
• Native hGH binds to both growth hormone and prolactin receptors

• rhGH  avoid un wanted side effects during therapy

• Site-specific mutagenesis of the cloned hGH : aa side chains that act

as ligands for Zn2+ (His-18, His-21, and Glu-174) ion required for the
high-affinity binding of hGH to the prolactin receptor

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 Optimizing Gene Expression Interleukin-3

Host system Promoter Expression Protein form

level (unit)
B.licheniformis Amylase 300 15 kD, mature

E coli LacZ 500 20 kD fusion

E coli lacZ 20 15 kD mature

Human cells Metallothionein 2 20-40 kD

Kluyveromyces lactis Lactase 20 20-100 kD

S cerevisiae Mating factor  20 20-100 kD

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Human, K lactis & S cerevisiae  able to glicasilate IL-3
How to alter amino acid ?

Site Directed Mutagenesis

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Oligonucleotide mutagenesis

• Used to alter a mutant Wild-

gene (and thus a Type
protein) in a sequence
desired way

Single-stranded template
DNA

Double-stranded
product transformed
into bacteria3/12/2019 Prof Sismindar PhD., Fak. Farmasi UGM, 2019 32
Oligonucleotide mutagenesis
• What is the role of a particular amino acid in the
function of the protein?
• What is the role of a particular base in the function of a
promoter?
• Engineering new functions into proteins.
• Removing unwanted functions from a protein
(glycosylation or phosphorylation sites).

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Delesi searah dengan ExoIII

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Mutasi terarah
• Fragmen disubklon pada M13
• Sintesis oligonukleotida mutan
• Hibridisasi, sintesis DNA untai ganda
• Isolasi mutan

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Siklus hidup M13

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Isolasi M13

Prof Sismindar PhD., Fak. Farmasi UGM, 2019 37

Prof Sismindar PhD., Fak. Farmasi UGM, 2019 38
Therapeutic Proteins from Recombinant Bacteria

Protein Function
 Protein DNase Application
digests DNA
cystic fibrosis
 Interferons
and stimulates cell growth treat different
Interleukins cancers; leukemia

 Superoxide
binds and destroys minimize tissue
dismutase
harmful free radicals damage after heart
attack

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 Antiviral
• First vaccine developed by Edward Jenner in 1796
• used a live cowpox virus to vaccinate humans against smallpox
• based on claims that milkmaids exposed to cowpox virus in
udder infections on cow never got smallpox disease.
• Exposure to cowpox fluid stimulated immune system of human
volunteers to develop protection against smallpox

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Antiviral Step 1: Binding
A virus consists of an outer envelope of
protein, fat and sugar wrapped around a set of
The HIV Life Cycle genes (in the case of HIV, genetic information
is carried as RNA instead of DNA) and special
enzymes.
HIV has proteins on its envelope that are
strongly attracted to the CD4+ surface receptor
on the outside of the T4-cell. When HIV binds
to a CD4+ surface receptor, it activates other
proteins on the cell's surface, allowing the HIV
envelope to fuse to the outside of the cell.

Step 2: Reverse Transcription

Step 3: Integration
Step 5: Translation

Step 6: Viral Assembly

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 DESAIGN FOR ANTIVIRAL

CD4

gp120

HIV
Th cell

CD4-toksin
Fusion prot

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GENETIC CONSTRUCTION of CD4-Pseudomonas EXOTOXIN A

Plasmid

T7 CD4 Exotoxin III

promoter RBS
Exotoxin II

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Selamat Belajar &
Thank You