Budiman, PROTEIN ENGINEERING, Budiman, Biomedik, 2014

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 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 STABILITY:
! Increased thermostability

 Increased activity in alternative pH
 Increased acitivity in different ionic strength
!  Improved protein folding
!  Decreased susceptibility to proteolysis
!  Pharmacokinetics
EXPRESSION:
 Improved expression levels in nonnatural
host
!  Targeted expression to different cellular
location
 Added tags to detect protein expression
 Altered posttranslational modification
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
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 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
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

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PROTEIN ENGINEERING

• Proteins are the workhorses of the cell.

The main thrusts in the field of protein engineering can be loosely

Traits that can be altered through protein

! - Addition of GST tag for purification using

! 2. DNA sequence that encodes the amino acid sequence,

Associated DNA Sequence

Site Directed Mutagenesis Combinatorial Mutagenesis

! 1. Make specific mutation ! 1. Make library of mutations

Newly engineered protein

Two main approaches for alteration of proteins:

Top down approach: !

1. Site Directed Mutagenesis !

Dapat juga dilakukan introduksi

 Kadang-kadang sulit diketahui mutasi seperti apa yang dapat

Saturation mutagenesis Error prone PCR

Metoda untuk memisahkan protein dengan sifat yang

2 Protokol diterapkan pada metoda kombinatorial:

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