Enzyme Technology

BCM5703

Dr. Mohd Shukuri Mohamad Ali

Email: shukuri@biotech.upm.edu.my
 Why purify?
By purifying a protein it can be clearly established that a particular
biological activity (enzymatic activity, signaling capacity, etc.)
actually resides in a unique protein.
Purified proteins serve as extremely valuable biochemical reagents
Determine mechanism (controlled, observable environment)
Structural determination
Sequence determination
Antibody production
Structure/function analysis - genetic engineering
Finding inhibitors
Detailed kinetic studies
Enzyme Production
 Cost of Purification
The effect of number of steps on the yield and costs
in a typical enzyme purification process. For example,

Specific Cost per Cost per

Step activity weight activity
Unit/mg ($/mg) ($/unit)

1 1 1.00

1 3 4 1.47
2 9 19 2.13
3 27 83 3.08
4 81 358 4.92
5 243 1536 6.32
 The basic techniques
Concentration (size) Electrophoresis
(size/charge)
precipitation
"native"
ultrafiltration denaturing
dialysis isoelectric focusing
centrifugation 2-dimensional
Chromatography Immunological
(size/charge/chemistry) (size/charge/chemistry)
ion exchange chromatography
size exclusion in situ imaging
affinity immunoblotting
 Guidelines for protein purification

Define objectives
Define properties of target protein and
critical contaminants
Minimize the number of steps
Use a different technique at each step
Develop analytical assays

Adapted from: Protein Purification Handbook. Amersham Biosciences. 18-1132-29, Edition AC

 How pure should my protein be?

Application Required Purity

Therapeutic use, in vivo
Extremely high > 99%
studies

Biochemical assays, X-ray

High 95-99%
crystallography

N-terminal sequencing,
antigen for antibody Moderately high < 95%
production, NMR
Separation of proteins based on
physical and chemical properties

Solubility

Binding interactions

Surface-exposed hydrophobic residues

Charged surface residues

Isoelectric Point

Size and shape

Basic scheme of protein purification

From: Protein Purification Handbook. Amersham Biosciences. 18-1132-29, Edition AC

Protein preparation, extraction,
clarification

Cell growth,
protein over-
expression
Cell lysis
Removal of
cell debris
Why use heterologous expression?

Proteins with low natural abundance

Site-directed mutagenesis

Protein engineering
Expression systems
Bacteria
Escherichia coli
Lactococcus lactis
other bacteria
Yeast
Pichia pastoris
Pichia methanolica
Saccharomyces cerevisiae
Insect cells
baculovirus
Mammalian cells
Cell Free
wheat germ extract
Escherichia coli extract
 Generation Time
Time required for cell to divide/for
population to double
Average for bacteria is 1-3 hours
E. coli generation time = 20 min
20 generations (7 hours), 1 cell becomes 1
million cells!
Standard Growth Curve
 Phases of Growth
Lag phase making new enzymes in
response to new medium
Log phase exponential growth
Desired for production of products
Most sensitive to drugs and radiation during
this period
 Phases of Growth
Stationary phase
nutrients becoming limiting or waste products
becoming toxic
death rate = division rate
Death phase death exceeds division
Expression System Characteristics
Characteristics E. coli Yeast Insect Mammalian
doubling time rapid (30 min) rapid (90 min) slow (18-24 hrs) slow (24 hrs)

cost of growth low low high high

medium
expression level high low high low high low moderate

protein folding refolding may be refolding may proper folding proper folding
required be required

N-linked glycos. no high mannose simple, no sialic complex

acid

O-linked glycos. no yes yes yes

phosphorylation no yes yes yes

acetylation no yes yes yes

acylation no yes yes yes

-carboxylation no no no yes

project cost low low middle high

 Inducible promoter systems

Promoter Regulation Inducer Expression Comments

T7 lacIq IPTG very high expensive induction
bacteriophage
trc (hybrid) E. lacI, lacIq IPTG moderately expensive induction
coli high
pL ( ) clts857 temp. shift moderately less likelyhood of leaky
to 42 C high uninduced expression

araBAD araC L-arabinose low to high fine-tune expression in

dose-dependent manner
 Expression of target protein
T7 RNA pol

. . . plasmid T7 RNA pol promoter Target protein plasmid . . .

Translation of
mRNA

No +
MW DinI IPTG IPTG

Target
protein
Protein isolation, concentration, and
stabilization

Reversible
precipitation
with salt or
organic
molecules
Fractional precipitation of proteins

Discard pellet
Precipitate
contaminants

Add Add Precipitant,

Centrifugation,
Precipitant, Chromatography
Discard supernatant,
Centrifugation Resuspend protein

Precipitate Discard
protein of supernatant,
interest Resuspend
protein
 Precipitation of proteins
by salting out

The ability of a salt to precipitate proteins

is described by the Hofmeister series:

_ _ _ _ _ _ _ _
Anions: SCN < ClO4 < NO3 < Br < Cl < acetate < SO4 2 < PO4 3

Cations: Na+ < K+ < NH4+

 Precipitation of proteins with
organic polymers

Adapted from: Protein Purification Handbook. Amersham Biosciences. 18-1132-29, Edition AC

Intermediate Purification

Liquid
chromatography
(lower resolution,
lower cost)
An introduction to liquid
chromatography
Protein solution applied to a
column

Column = solid porous matrix

(stationary phase) + liquid (mobile
phase)

Proteins separated based on

differing interactions with stationary
and mobile phases

Mobile phase conditions can be

adjusted to increase or decrease
affinity of protein for stationary
phase (gradient)
Fractionation during
chromatography

Proteins separated
by chromatography
are collected in
fractions to keep
them separated
Equipment for liquid chromatography

KTA FPLC Refrigeration

Buffer reservoirs
Gradient maker
Way to apply
buffers and protein
sample to column
Column
Detection system
Fraction collector
Controller /
Recorder
Sequence of a general
chromatography run
 Changing buffer between
chromatography steps: Dialysis

Need porous membrane with

specific molecular weight cutoff
(MWCO)
Proteins stay inside membrane
Molecules smaller than MWCO
free to equilibrate across
membrane

Generally consists of 3, 2-hour

steps

Rule of thumb: volume of

solution to change into > 100x
volume of protein solution
 Types of liquid chromatography

Adsorption Chromatography
Proteins bind to stationary phase
Proteins eluted by altering mobile phase
Includes: affinity, hydrophobic interaction, ion exchange, and
chromatofocusing

Solution Phase Chromatography

Proteins do not bind to stationary phase
Progress of proteins through column impeded by matrix of
stationary phase
Includes: size exclusion chromatography (aka gel filtration)
Size exclusion chromatography

Smaller Proteins

Bigger Proteins
Affinity Chromatography
 Affinity Chromatography

Most commonly-used adsorption chromatography

technique

Can be used on protein with natural ligands

Often involves covalent attachment of affinity tag to

protein

Because of unique tag, provides rapid, specific

cleanup in one chromatography step*

Can allow for automation of protein purification

 Popular Small Affinity Tags

Tag Matrix Eluting Residues Sequence Notes

Agent
His Ni2+-NTA, imidazole or 5-15 HHHHH native or
Talon, Cobalt low pH denaturing;
inexpensive
resin
FLAG anti-FLAG low pH or 8 DYKDDDDK high
antibody EDTA/EGTA specificity;
harsh elution
cond.
Strep II modified desthiobiotin 8 WSHPQFEK expensive
streptavidin resin

S- S-protein enzymatic 15 KETAAAKFERHMDS detection

peptide cleavage possible;
expensive
resin
 Popular Large Affinity Tags

Tag Matrix Eluting Size Notes

Agent
MBP amylose maltose 40 kDa solubility + secretion
enhancer;
inexpensive resin
GST glutathione reduced 26 kDa inexpensive resin
glutathione

cellulose-binding cellulose water or 4-20 kDa secretion enhancer

domains GdHCl

calmodulin-binding calmodulin EDTA/EGTA 4 kDa detection possible;

peptide expensive resin

His-patch Ni2+-NTA, Talon imidazole 11.7 kDa solubility + S-S

thioredoxin enhancer
MBP tagged protein

www.2010.igem.org
 Which Tag to Use?
Specificity of binding interaction

Cost of resin

Native vs. denaturing elution

Presence of metals

Expression level, solubility & toxicity of target protein

Tag removal
 Tag Removal

NH2 tag linker protein

considerations:

effect on structure
effect on function
DDDDK flexibility
protease
protein 1 sequence
 Tag Removal

Excision Site Cleavage Enzyme Notes

D-D-D-D-K X enterokinase active: pH 4.5-9.5, 4-45 C

X cannot be P
secondary cleavage sites
I-D/E-G-R X factor Xa protease X cannot be P/R
secondary cleavage sites
L-V-P-R G-S thrombin biotynilated form available
secondary cleavage sites
E-N-L-Y-F-Q G TEV protease active: wide range of T
His-tagged form available
L-E-V-L-F-Q G-P PreScissionTM protease engineered with GST tag
 Hydrophobic interaction
chromatography (HIC)
Hydrophobic group bound to solid phase
Binding
high salt (increases water surface tension, decreases
available water molecules, increases hydrophobic
interactions)
Elution
decrease salt
add detergent
decrease polarity
of mobile phase
 Polishing steps

Liquid
chromatography
(higher resolution,
higher cost)

From: Protein Purification Handbook. Amersham Biosciences. 18-1132-29, Edition AC

 Size Exclusion

Conductivity
Sephacryl S-100
Load in Tris buffer + 200 mM KCl
UV
Elute with Tris buffer + 200 mM KCl

Increasing [KCl] in % of total buffer

% buffer w/
200 mM KCl
Increasing Abs @ 280 nm

Increasing [Salt]
Increasing Volume and Fraction #
 Types of liquid chromatography

Adsorption Resin Chemical Equilibrate Elution

Type Separation By Names of Resins
or Solution Group With With
Metal, Ig,
Low High Hydroxyapatite,
Affinity Adsorption Specific Ligand Ligand Binding
[Ligand] [Ligand] Heparin Sepharose,
Any ligand
Butyl sepharose, Octyl
Hydrophobic Hydrophobic Hydrophobic
Adsorption High Salt Low Salt sepharose, Phenyl
Interaction Groups Effect sepharose
Positively charged Coulombic Mono-Q, Source-Q,
Anion Exchange Adsorption Low Salt High Salt
ions Interacions DEAE

Negatively charged Coulombic Mono-S, Source-S,

Cation Exchange Adsorption Low Salt High Salt
ions Interacions CM

Negatively charged pH
Chromatofocusing Adsorption Isoelectric Point Poly-buffer Mono-P
ions gradient
Size Exclusion Solution Size / Shape of Sephacryl #,
Pores Same Buffer
(gel filtration) Phase Protein Sephadex #
 Liquid chromatography techniques
advantages and disadvantages

Type of
Advantages Disadvantages Resolution
Chromatography
Resins and ligands
Affinity Quick and specific
can be expensive Low to Medium
Can be used directly Relatively low
Hydrophobic
from ammonium resolution and binding Low to Medium
Interaction sulfate precipitation capacity

Protein solution must

Ion Exchange Versatile resin choices
start at low [salt] Medium to High

pH gradient can be
Chromatofocusing High resolution
harsh for protein High
Distinct from other
techniques, Can be
Size Exclusion used analytically or for
Long run time Low to High
buffer exchange
 Protein detection methods

SDS-PAGE
Visual confirmation

UV Spectrophotometry
Absorbance @ 280 nm
Due mostly to Trp

Colorimetric Techniques
Color change proportional to [protein]
Bradford, Lowry, etc

J.S.C. Olson and John Markwell. Current Protocols in Protein Science (2007) 3.4.1-3.4.29
Electrophoresis

Tris-glycine buffer
10% SDS
Final steps in purification
Check purity by detection methods
Test for interfering contaminants
Nucleases
Proteases
Toxins

Concentrate your protein

Precipitation
Centricons
Small column with high binding capacity

Choose a storage buffer and storage conditions

Consider intended use of protein
Stabilizing additives
Flash freeze protein and store at -80o C

Confirm identity of purified protein

Mass spectrometry
N-terminal sequencing
Analytical assays
Basic scheme of protein purification

Cell growth, Liquid

protein over- chromatography
expression Reversible (lower resolution,
precipitation lower cost)
Cell lysis with salt or
Removal of organic
cell debris molecules

Liquid
chromatography
(higher resolution,
higher cost)
Guidelines for protein purification

Define objectives
Define properties of target protein and
critical contaminants
Minimize the number of steps
Use a different technique at each step
Develop analytical assays

Adapted from: Protein Purification Handbook. Amersham Biosciences. 18-1132-29, Edition AC

Common additives and their uses

Adapted from: Protein Purification Handbook. Amersham Biosciences. 18-1132-29, Edition AC

Precipitation with (NH4+)2SO42- based
on % saturation