PROTEIN ENGINEERING
Budiman Bela
OUTLINE
Introduction
Protein Engineering Goals
Preliminary Requirements
Rational Mutagenesis and Protein Design
Combinatorial Methods
Assessment of Improvements and Cycle Repetition
Conclusions
INTRODUCTION
• Proteins are the workhorses of the cell.
• There are 20 common amino acids that allow different
combinations and formation of proteins with various functions and
capabilities:
1. Specific binding of ligands
2. Catalysis of complex chemical reactions
3. Functionality in extreme environments
4. Transportation of valuable molecules
5. The exhibition of diverse structural and material
properties
INTRODUCTION
The main thrusts in the field of protein engineering can be loosely
divided into two areas:
1. Investigation and verification of hypotheses during the study of
protein functions
2. Engineering of proteins for desired improvements
PROTEIN ENGINEERING GOALS
Traits that can be altered through protein
engineering:
! Activity
! Stability
! Expression
! Other traits
PROTEIN ENGINEERING GOALS
PROTEIN ACTIVITY:
Improved catalysis with natural substrate or
cofactor
Improved catalysis with nonnatural
substrate or cofactor
! Increased catalysis in nonnatural solvent
! Improved ligand binding
! Decreased effects of inhibition
PROTEIN ENGINEERING GOALS
PROTEIN STABILITY:
! Increased thermostability
Increased activity in alternative pH
Increased acitivity in different ionic strength
! Improved protein folding
! Decreased susceptibility to proteolysis
! Pharmacokinetics
PROTEIN ENGINEERING GOALS
EXPRESSION:
Improved expression levels in nonnatural
host
! Targeted expression to different cellular
location
Added tags to detect protein expression
Altered posttranslational modification
PROTEIN ENGINEERING GOALS
OTHER TRAITS:
Added tags to facilitate purification
Altered tendency for polymerization
Added tags to visualize localization
Engineered allosteric binding sites
Altered isoelectric point
! Decreased immunogenicity
Addition of tag to facilitate purification:
! - Addition of GST tag for purification using
glutathione-sepharose collumn
! - Addition of histidine tag for purification using nickel
collumn
PRELIMINARY REQUIREMENTS
Before a protein engineering project can begin, a certain
amount of information is required about the protein of interest:
! 1. Amino acid sequence of the protein
! 2. DNA sequence that encodes the amino acid sequence,
(Amplification of DNA for sequencing PCR) cloning in
plasmid
3. Although not required, information about the the 3-
dimensional (3-D) structure of a protein can aid the
engineering of a protein
PRELIMINARY REQUIREMENTS
Protein to be engineered
Associated DNA Sequence
Site Directed Mutagenesis Combinatorial Mutagenesis
! 1. Make specific mutation ! 1. Make library of mutations
! 2. Assay effect of mutation on ! 2. Screen or select for improved
protein function proteins
! 3. Repeat as necessary ! 3. Determine specific mutations
! 4. Repeat as necessary
Newly engineered protein
Mutagenesis of Proteins
Two main approaches for alteration of proteins:
! Rational mutagenesis
! Combinatorial Methods
Rational Mutagenesis
Top down approach: !
A hypothesis is made about mutations at a
specific location (often guided by 3-D structural
information) the hypothesis is tested
through the mutation of specific smino acids
and assays of the subsequent mutant proteins
Combinatorial mutagenesis
Bottom up approach: !
A library of different mutant proteins is
produced a method is then developed to
screen or select members of the library that
have an improved trait, the mutations that
caused the improvement are determined later
Rational Mutagenesis
1. Site Directed Mutagenesis !
2. Other methods:
1. Use of restriction sites
2. Introduction of restriction site by site !
directed mutagenesis (restriction sites
can be introduced without altering the
associated protein sequence) cut and paste of DNA
sequences: fusion proteins, swap domains of proteins,
remove entire section of proteins
Combinatorial method
dengan
Teknik DNA shuffling
Site Directed Mutagenesis
(penyambungan domain-domain
dari spesies enzim yang berbeda
untuk menghasilkan enzim baru
dengan sifat-sifat dari setiap
spesies sesuai dengan fragmen
gen yang membentuk gen enzim
baru
Dapat juga dilakukan introduksi
situs restriksi untuk menyisipkan
fragmen DNA dengan sifat tertentu:
Contoh: NLS viruses
Site Directed Mutagenesis
Site Directed Mutagenesis
Site Directed Mutagenesis
Site Directed Mutagenesis
Site Directed Mutagenesis
Metoda Kombinatorial
Kadang-kadang sulit diketahui mutasi seperti apa yang dapat
menghasilkan sifat tertentu pada suatu protein
Misalnya, untuk meningkatkan termostabilitas suatu protein
masih sulit untuk dilakukan hanya dengan melakukan prediksi
pada struktur 3 dimensi (3-D) untuk menentukan asam amino
mana yang menentukan sifat ini
Dengan metoda ini dapat dilakukan mutasi pada berbagai
tempat untuk kemudian dilakukan identifikasi protein mutan
yang mana yang dapat menghasilkan fungsi yang diinginkan
Metoda
Kombinatorial
KONSTRUKSI PUSTAKA
KOMBINATORIAL
Metoda KONSTRUKSI PUSTAKA
Kombinatorial KOMBINATORIAL
Saturation mutagenesis Error prone PCR
Gene Shuffling
Metoda
Kombinatorial
KONSTRUKSI PUSTAKA
KOMBINATORIAL
Metoda
Kombinatorial
KONSTRUKSI PUSTAKA
KOMBINATORIAL
Metoda Kombinatorial
Metoda untuk memisahkan protein dengan sifat yang
diinginkan dan mengidentifikasi mutasi penyebab
perubahan:
1. Penelusuran (Skrining): semua pustaka yang ada
diperiksa secara acak untuk mencari protein dgn
sifat yang diinginkan
2. Metoda seleksi: Pustaka diseleksi sedemikian rupa
sehingga hanya yang memiliki perbaikan sifat yang
ditelusuri
Metoda Kombinatorial
2 Protokol diterapkan pada metoda kombinatorial:
1. Metoda pembuatan pustaka kombinatorial dengan
keragaman yang tinggi sehingga meningkatkan
kemungkinan diperoleh protein dengan sifat yang
diinginkan
2. Penentuan metoda yang digunakan untuk memi-
sahkan protein dengan sifat yang diinginkan dan
mengidentifikasi mutasi penyebab perubahan
PENILAIAN PERBAIKAN SIFAT
PROTEIN
Pendahuluan
Tujuan Rekayasa Protein
Mutagenesis Protein
Metoda Kombinatorial
Penilaian perbaikan sifat protein
Kesimpulan
TERIMAKASIH
