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An Introduction to Protein Puri!cation:

Methods, Technologies and
Applications

Advanced protein puri!cation

techniques enhance protein activity and
yield, facilitating breakthroughs in
research.

Article Published: July 5, 2024

| Héctor Zamora Carreras, PhD

Credit: iStock

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Today, the ability to obtain pure proteins is essential

for developing targeted drugs, creating vaccines and
understanding biological processes at a molecular
level. The development and optimization of protein
purification methodologies have become increasingly
important to meet the high demand for these
proteins, ensuring they are free from contaminants
that could a!ect their function.

Advanced purification techniques enhance the yield

and activity of proteins, facilitating breakthroughs in
medical research and industrial applications.
Continuous improvements in these methodologies
enable more e"cient, cost-e!ective and scalable
production of high-quality proteins, driving
innovation and discovery in numerous scientific
domains.

Contents

What is protein purification?

What is a typical protein purification

protocol?

- Sourcing the protein

- Extraction

- Solubilization and stabilization

- Purification

- Characterization and analysis

Protein extraction methods

- Mechanical methods

- Non-mechanical methods

Protein purification techniques

- Centrifugation

- Precipitation

- Chromatography

- Ultrafiltration and dialysis

- Electrophoresis

Recombinant protein expression

Purification of recombinant proteins

- 1. Protein tagging and a"nity purification

- 2. Solubilization/refolding

Protein analysis

- Protein concentration determination

- Purity analysis

- Activity assays

- Structural analysis

- Stability testing

- Quantitative analysis

References

What is protein purification?

Protein purification is a process in molecular biology

and biochemistry that involves isolating a specific
protein from a complex mixture, often sourced from
cells, tissues or other biological materials.1,2 This
process is essential in many fields including:

Biological research: to develop reagents like

enzymes and antibodies that can be used as
molecular biology tools for understanding
cellular processes.3
Diagnostics: purified proteins are used to
develop assays and tests for diseases.4
Environmental monitoring: protein-based
biosensors are used to detect contaminants.5
Food and cosmetics: protein content in food
and cosmetic products must fulfill certain safety
standards due to the risk of allergic reactions.6
Forensic science: utilizes proteins for
identifying substances in criminal
investigations.7
Biopharmaceutical development: purified
proteins are pivotal for drug development and
production, including therapeutic proteins and
vaccines.8

What is a typical protein purification

protocol?

A typical protein purification workflow involves

several essential steps to isolate and purify the
protein of interest while minimizing contaminants
and maximizing yield and activity (Figure 1). This
structured approach ensures the e!cient isolation
and detailed analysis of proteins.

Sourcing the protein

Proteins can be isolated from native tissues or cells

where they are naturally expressed. This approach is
often used for proteins that are di!cult to express
recombinantly or when studying proteins in their
native context. Alternatively, many proteins are
produced using genetically engineered organisms
(see “Recombinant protein expression” section),
allowing for high yields and easy manipulation of the
expression.1,8

Extraction

The goal of this step is to break open cells to release

their contents, including the target protein. It can be
achieved by di"erent approaches, such as
mechanical disruption (commonly used for bacterial
and yeast cells), chemical disruption (using
detergents, organic solvents or chaotropic agents)
and enzyme disruption (which can help break down
cell walls, especially in bacterial cells). Freeze-thaw
cycles and pressure cycling are alternative methods
to lyse cells e"ectively. For further details, see the
“Protein extraction methods” section. 1,8

Solubilization and stabilization

This step ensures that proteins remain soluble and

stable in solution. This is achieved by employing
appropriate bu"ers, protease inhibitors to prevent
degradation and other agents to maintain protein
solubility. For membrane proteins, detergents may
be required to avoid their aggregation and/or
precipitation. 1,8

Purification

Purification involves the isolation of the target

protein from other cellular components. Some of the
main techniques used for this goal are: i)
Centrifugation: to separate cellular debris. ii)
Precipitation methods: to concentrate proteins by
altering solubility. iii) Chromatographic techniques
(a!nity, ion exchange, size exclusion, and
hydrophobic interaction): to separate proteins based
on di"erent properties (molecular size, charge etc.).
iv) Ultrafiltration and dialysis: to concentrate and
desalinate samples, which is essential for
downstream applications. For further details, see the
“Protein purification techniques” section. 1,8

Characterization and analysis

This step is necessary to confirm the identity, purity

and functionality of the purified protein. Some of the
most common methods for the characterization and
analysis of purified proteins are: i) Electrophoresis
(Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE)), which assesses purity
and molecular weight. ii) Spectroscopic methods (UV-
Vis, fluorescence), which determine concentration
and structural properties. iii) Activity assays, which
verify the functional integrity of the isolated protein.
iv) Advanced techniques like nuclear magnetic
resonance (NMR) spectroscopy, X-ray crystallography
and mass spectrometry which provide detailed
structural and functional insights. 1,8

Figure 1: Workflow of a typical protein purification

procedure. Credit: Technology Networks.

Protein extraction methods

Protein extraction is a crucial step in the purification

process, involving the disruption of cells to release
proteins. Various methods are used depending on
the type of cells and the stability of the target protein
(Figure 2). Each method has its advantages and
limitations, which are discussed below:

Mechanical methods

Skip to
Homogenization: This technique content or Skip
employs
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mechanical shear force to break open cells. It's highly
e"ective for tough plant and animal tissues and it is
scalable. However, the process can generate heat,
which may denature sensitive proteins.9

Sonication: Utilizes ultrasonic waves to disrupt cell

membranes. This is a quick approach commonly
used for bacterial cells and it is very e"ective for
small volumes. It requires cooling to prevent protein
denaturation due to the generated heat.1

Pressure cycling: Consists of using high pressure to

induce cell lysis and is appropriate for tough cells like
yeast, being gentle on proteins. The disadvantage of
this methodology is that it requires specialized
equipment (a barocycler) which is not widely
available.10

Non-mechanical methods

Detergents: Solubilize cell membranes by disrupting

lipid bilayers. Some common detergents include
Triton X-100 and SDS. This process is easy to use and
very e"ective for membrane proteins, but it can
cause denaturation
Analysis if used at high concentrations.
& Separations SubscribeIt
may require removal before further purification. 1,8
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Organic
Home solvents: Solvents such
Analysis & Separations as ethanol
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Content

acetone are used to precipitate proteins and disrupt

membranes. It is a quick approach, but it is not
suitable for all protein types, as it can cause
denaturation. 1,8

Chaotropic agents: Disrupt the hydrogen bonding

network in proteins, aiding in solubilization.
Examples include urea and guanidine hydrochloride.
This approach is especially useful for insoluble
proteins, but can denature proteins and often
requires subsequent refolding steps. 1,8,11

Freeze-thawing: This procedure involves repeated

cycles of freezing and thawing to lyse cells by ice
crystal formation. It is a simple way to lyse cells since
it doesn’t require special equipment. However, it is
time-consuming and not very e!cient for some cell
types. 1,8

Enzymatic treatment: Sometimes enzymes, such as

lysozymes, are used to break down bacterial cell
walls, in combination with other methods for
enhanced e!ciency. This approach is very specific
and maintains protein integrity. Unfortunately, its
use is limited to bacterial cells and requires
additional steps for a complete cell lysis. 1,8

Each extraction method o!ers distinct advantages

and limitations. The choice of method depends on
the type of cells being used, the properties of the
target protein and the subsequent purification steps
planned.

Figure 2: Commonly used protein extraction

methods. Credit: Technology Networks.

Protein purification techniques

Protein purification involves several techniques, each

tailored to isolate proteins based on specific
properties like size, charge or a!nity. Here’s a
detailed look at each method:

Centrifugation

Centrifugation separates components based on their

density by spinning samples at high speeds. During
the process, heavier particles, such as cellular debris,
sediment at the bottom, allowing the lighter
supernatant, which contains the proteins, to be
collected. This technique is often the first step in
purification, used to remove large contaminants and
concentrate proteins from crude extracts. 1,12

Precipitation

Precipitation involves altering the solubility of

proteins to cause them to aggregate and precipitate
out of solution. This can be achieved using salts
(salting out), such as ammonium sulfate, organic
solvents or changes in pH. Although this process can
lead to the co-precipitation of contaminants, it is
useful for the initial protein concentration and
fractionation, particularly when working with large
volumes. 1,13

Chromatography

Chromatography comprises of a varied set of

versatile and widely used purification techniques that
can be used to separate proteins based on various
properties (Figure 3).

A!nity chromatography exploits specific

interactions between a protein and a ligand attached
to a resin. The target protein binds to the ligand,
allowing other components to be washed away,
followed by elution of the protein under specific
conditions. This technique is suitable for proteins
with known binding partners or tags (e.g., His-tagged
proteins). 1,14

Ion exchange chromatography separates proteins

based on their charge. Proteins bind to charged
resins (cationic or anionic) and are eluted by
increasing the ionic strength or changing the pH of
the bu"er. This is useful for proteins with well-
defined charge, and it is often used as an
intermediate purification step. 1,15

Size exclusion chromatography (SEC) separates

proteins based on size by passing them through a
column filled with porous beads. Smaller proteins
enter the pores and elute later, while larger proteins
bypass the pores and elute earlier. This technique is
usually used for separating monomers from
aggregates and as a final polishing of the protein
purification process. 1,16

Hydrophobic interaction chromatography

(HIC) separates proteins based on hydrophobicity.
Proteins bind to hydrophobic groups on a resin at
high salt concentrations and elute as the salt
concentration decreases. This approach is
appropriate for proteins containing highly
hydrophobic domains and helps remove undesired
aggregates. 1,17

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Figure 3: Scheme of the columns employed in some

of the typical chromatography techniques used for
protein purification. Credit: Technology Networks.

Ultrafiltration and dialysis

Ultrafiltration is a methodology that uses

semipermeable membranes to concentrate and
desalt protein solutions by applying pressure. 1,18 On
the other hand, dialysis is a process that involves
placing the protein solution inside a membrane with
selective permeability, allowing small molecules to
di"use out to a surrounding solution. These
methodologies are used to remove small
contaminants, exchange bu"ers and concentrate
proteins. 1,19

Electrophoresis

Electrophoresis separates proteins through a gel

matrix based on size and charge by applying an
electric field. One of the most common variants is
SDS-PAGE, which uses a detergent (SDS) to denature
proteins and separate them by size through a
polyacrylamide gel matrix.20 Electrophoresis is both a
technique for protein separation and the analytical
assessment of protein purity and molecular weight
determination.1

By understanding the strengths and limitations of

each technique (see Table 1), researchers can choose
the most appropriate methods for their specific
protein purification needs, ensuring high purity and
functionality of the target protein.

Table 1: Summary of protein purification techniques

with their strengths, limitations and the most
suitable samples.

oll bar at the bottom of the table to view more.

Strengths Limitations Suitable samples

Simple, cost- Not highly Initial separation,

e"ective, selective, may crude extracts
scalable not remove
small
contaminants

Cost- Can co- Initial concentration,

e"ective, precipitate fractionation
rapid, contaminants,
simple requires
optimization

High Ligand Tagged proteins,

hy specificity binding needs specific binding
and purity optimization, partners
expensive
resins

High Requires Proteins with defined

hy resolution, careful charge properties
scalable pH/ionic
strength
control

Gentle, Lower Final polishing,

hy useful for resolution, separating
desalting limited monomers/aggregates
and bu"er capacity
exchange

E"ective for Requires salt Hydrophobic proteins,

hydrophobic optimization, aggregate removal
hy proteins may denature
proteins

Simple, non- Limited by Concentration, bu"er

denaturing, membrane exchange
cost- pore size
e"ective

s High Typically, not Purity assessment,

resolution, preparative, molecular weight
analytical can denature determination
proteins

Recombinant protein expression

Recombinant proteins are genetically engineered to

be produced by host cells that have been
transformed with recombinant DNA. This DNA
encodes the protein of interest, allowing the host
cells to produce the protein in large quantities. The
process of expressing recombinant proteins involves
several steps (Figure 4):21,22

1. Gene cloning: The gene encoding the protein

of interest is isolated and inserted into a
plasmid or other vector. This recombinant DNA
construct often includes regulatory elements
such as promoters and terminators to ensure
proper expression.
2. Transformation/transfection: The
recombinant DNA is introduced into host cells.
For bacterial and yeast cells, this process is
called transformation, while for mammalian
and insect cells, it is known as transfection.
3. Selection: Host cells that have successfully
incorporated the recombinant DNA are selected
using antibiotic resistance markers or other
selection systems encoded on the vector.
4. Expression: The selected host cells are cultured
under conditions that induce the expression of
the recombinant protein. This step may involve
optimizing growth conditions such as
temperature, pH and nutrient supply.
5. Harvesting: The host cells are lysed to release
the recombinant protein, which is then purified
using the aforementioned techniques.

Figure 4: Workflow of a typical recombinant protein

expression protocol. Credit: Technology Networks.