This document provides an overview of protein expression in mammalian cells and protein purification techniques. It discusses how mammalian cells are the preferred system for producing mammalian proteins due to their ability to perform post-translational modifications. Common mammalian cell lines for expression include HEK293 and CHO cells. Plasmid vectors are typically used to insert the gene of interest, and transfection methods like lipofection and electroporation can be used to introduce the plasmid into cells. Affinity, size-exclusion, and ion-exchange chromatography are common protein purification techniques.
2. A mammalian host system is the preferred expression
platform for producing mammalian proteins that have the
most native structure and activity.
Mammalian expression is the system of choice for
studying the function of a particular protein in the most
physiologically relevant environment, because it allows for
the highest level of posttranslational processing and
functional activity of the protein.
They have been successfully applied in the
biopharmaceutical production of cytokines, monoclonal
antibodies, growth factors and so on.
For mammalian cell expression , the gene of interest
insert through the vector based method and transfection
methods.
PROTEIN EXPRESSION IN MAMMALIAN
CELLS
3. 2 TYPES OF MAMMALIAN CELLS COMMONLY
USED
HEK293 (Human
Embryonal Kidney 293)
Cells
• Human Embryonic Kidney
293 (HEK293) cells are a
specific cell line originally
derived from human
embryonic kidney cells
grown in tissue culture.
• They are very easy to grow
and transfect very readily,
thus widely used in cell
biology research and
biotechnology industry.
• Transient expression
CHO (Chinese Hamster
Ovary) cells
• Chinese hamster ovary
(CHO) cells are a cell line
derived from the ovary of
the chinese hamster.
• They’re often used in
biological and medical
research.
• Transient and stable
expressions
4. 2 TYPES OF CELL EXPRESSION
Inducible
Expression
• An inducible promoter allows controlling the timing of
gene expression. In the absence of the inducer, the
gene is not expressed.
• This option is ideal for expressing toxic proteins.
• For regulated and inducible expression of the gene of
interest, use the Invitrogen T-REx Expression System,
Invitrogen Flp-In T-REx system.
Constitutive
Expression
• if working with a nontoxic gene and the timing of
expression is not important, choose an expression
vector with a constitutively expressing promoter.
• For this purpose, use the pcDNA vectors.
• pcDNA vectors are available with either the CMV,
EF-1, or UbC promoter, a variety of different epitope
tags, standardized detection or purification of
proteins, several selection markers, and different
cloning formats.
5. PLASMID BASED EXPRESSION
VECTOR
Expression vector is a vector that allows the
transcription and translation of a foreign gene inserted
into it.
Eukaryotic expression vector is a shuttle vector, containing ColE1 ori,
SV40 origin and has an efficient promoter (e.g. SV40 early promoter,
Rous Sarcoma virus promoter, adenovirus major late promoter, or
human cytomegalovirus promoter).
In most cases, first attempts to transiently express recombinant
proteins were executed with ‘standard’ expression vectors that
contain strong viral promoters, such as SV40 or a CMV promoter
from cytomegalovirus (CMV).
More recently, at least one non-viral promoter, the
elongation factor (EF-1) promoter. It is appears to be
as strong or stronger than some viral promoters.
6.
7. TRANSFECTION
Transfection is defined as the process of inserting nucleic
acids (e.g., plasmid DNA, cDNA, mRNA, miRNA, siRNA)
into the eukaryotic cells. In addition, proteins and
nanoparticles such as beads or dyes can be transfected.
Transfection is an important and widely used tool for
analyzing the function of various genes and used for
gene silencing via RNAi, gene editing via CRISPR/Cas9,
as well as over expression studies by cellular integration
of plasmid DNA, mRNA, or proteins.
Infection:- viral-mediated process in which target cells are
infected with a virus carrying cloned DNA sequence in its
genome
8. TYPE OF TRANSFECTION
STABLE
• Viral and Microinjuction
based
• Long term Expression
• Foreign gene non-
specific integrated into
genome
• Complex process
TRANSIENT
• Chemical and
Electroporation based
• Short Term Expression
(24-96hr)
• Foreign gene not
integrated into genome
• Easy process
11. PHYSICAL METHODS
The target cell is positioned under a microscope
and being fixed by a pipette then the nucleotide
solution is then directly injected into the
cytoplasm and/or the nucleus using a fine glass
capillary needle.
However, as each single cell needs to be
microinjected individually, this method is very time-
consuming, expensive but highly effective.
Microinjection is mostly used for single cell
manipulation or the generation of transgenic animals.
1.
Microinjuction
12.
13. During electroporation, a mixture of the cells and the
nucleotide of interest is exposed to an intense electric field.
This leads to transient cell membrane destabilization,
making the cell membrane permeable to the nucleotides that
are present in the surrounding solution, to enter the
cytoplasm.
After removing the electric field, the cell membrane stabilizes,
enclosing the nucleotides in the cytoplasm, where they are
expressed.
Electroporation allows for the transient and stable
transfection of any cell type.
This method is easy and reliable, but it requires high cell
numbers due to high rates of cell death during the procedure.
Therefore, electroporation is not suitable for sensitive and
difficult-to-culture cell types, such as primary cells.
2. Electroporation
14.
15. CHEMICAL METHODS
A mixture of neutral and cationic liposomes forms complexes with the
nucleotides of interest. These complexes pass the cell membrane and
then release the nucleotides into the cytoplasm via endocytosis. Then,
escape the endosome or undergo lysosomal degradation. After
successful endosomal escape, the nucleotide is expressed in the
target cells.
Lipofection is commonly used to transfer nucleic acids such as RNA or DNA into
eukaryotic cells.
This method is easy to apply and yields highly reproducible results of transient and
stable transfections.
However, lipofection efficiency strongly depends on the cell type and has to be
tested and optimized in advance.
Especially for primary and non-dividing cells, the viability after the transfection
process might be decreased due to the high cellular sensitivity.
1.
Lipofe
ction
16.
17. 2. CALCIUM PHOSPHATE
A mixture of the nucleotides, calcium, and phosphate buffer
forms a precipitate that is taken up by the cells via
endocytosis. Then, the nucleotides either escape the
endosome or undergo lysosomal degradation.
The calcium phosphate transfection is an insoluble precipitate
with DNA and for both transient and stable transfection. It is an
inexpensive and simple method for transient or stable
nucleotide transfer into most cell lines.
The transfection efficiency, strongly depends on the cell
constitution, the pH, and the quality and the amount of the
used nucleotides.
The calcium phosphate transfection is toxic and therefore not
suitable for most sensitive primary cell lines.
18.
19. 3. CATIONIC POLYMERS
The negatively charged nucleotide backbones form complexes
with cationic polymers, such as diethylaminoethyl (DEAE)-
dextran. The complex is then taken up by the cells, mostly via
endocytosis. If no lysosomal degradation occurs, the nucleotide
can escape from the endosome into the cytosol of the host cells,
subsequently resulting in transgene expression.
Using cationic polymers, nucleotides can be transiently
transfected into eukaryotic cells in an inexpensive and simple
manner.
As a drawback, transfection by cationic polymers has a very low
efficiency (<10%) in a number of cell types, including primary
cells.
Further, cationic polymers are highly cytotoxic and therefore not
suitable for transfection of sensitive cells and generation of stable
cell lines.
20.
21. VIRUS BASED MAMMALIAN
TRANSFECTION
Adenoviruses are a class of double stranded DNA viruses that efficiently
deliver nucleotides directly into target cells. The virus enters the host cell
via endocytosis. Following endosomal escape, the viral genome is
transported into the nucleus, where it is expressed by the replication
machineries of the host cell.
Nowadays, replication-deficient adenoviruses are widely used for
transduction and gene therapy, due to their high efficiency and low
pathogenicity.
Adenoviral vectors have proven to be a very successful transduction tool
in many eukaryotic cell types, such as human and rodent cells. Besides
dividing cell lines, this method gives access to difficult-to-transfect cells,
such as primary cells.
Adenovirus-mediated transduction is always transient, meaning that no
nucleotide integration into the host genome occurs. Transduction
efficiencies of up to 100% can easily be achieved.
1. Adenoviral
Transduction
22.
23. 2. LENTIVIRAL
TRANSDUCTION
Lentiviruses a subclass of retroviruses have the ability to
permanently integrate into the genome of the host cell. After
the virus has entered the cell, the viral RNA is transcribed by
the reverse transcriptase to produce double-stranded DNA
that enters the nucleus. Finally, the transgene is integrated
into the host genome via the lentiviral integrase enzymes.
Lentiviruses enable stable gene transfer in vitro and in vivo, as
they integrate into the host cell genome and offer the
possibility of positive cell selection.
They have a broad host cell range allowing transduction of
many cell types such as primary neurons, lymphocytes, and
macrophages. Moreover, lentiviral vectors have also proven to
be effective in transducing brain, liver, muscle, and retina in
vivo without toxicity or immune responses.
24.
25. COMMONLY USED TECHNIQUES FOR
PROTEIN PURIFICATION
1. Affinity
Chromatography
2. Gel
permeation
3. Ion Exchange
Chromatography
The most common methods for preparative
purification of proteins all involve chromatography.
26. 1. AFFINITY
CHROMATOGRAPHY
Affinity Chromatography is a separation technique based upon
molecular conformation.
A ligand which can make a complex with specific protein (dextran,
polyacrylamide, cellulose etc) binds the filling material of the
column through the spacer arm.
The specific protein which makes a complex with the ligand is
attached to the solid support (matrix), and retained in the column,
while free proteins leave the column.
The target protein leaves the column by means of changing its
ionic strength through alteration of pH or addition of a salt solution.
This chromatography technique is used for the purification of
enzymes, hormones, antibodies, nucleic acids, and specific
proteins.
27.
28. 2. GEL- PERMEATION (MOLECULAR SIEVE)
CHROMATOGRAPHY
The basic principle of this method is to use dextran containing materials to
separate macromolecules based on their differences in molecular sizes.
In a gel- permeation column stationary phase consists of inert molecules with
small pores. The solution containing molecules of different dimensions are
passed continuously with a constant flow rate through the column.
Smaller Molecules cannot permeate into gel particles, and they are retained
between particles within a restricted area while Larger molecules pass through
spaces between porous particles, and move rapidly through inside the
column. As a result, Firstly larger molecule elute out from column, then the
retained smaller molecules eluted later.
This procedure is basically used to determine molecular weights of proteins,
and to decrease salt concentration of protein solutions.
Sephadex-G type is the most frequently used column material. Besides,
dextran, agorose, polyacrylamide are also used as column materials.
29.
30. 3. ION- EXCHANGE
CHROMATOGRAPHY
Ion- exchange chromatography is based on electrostatic
interactions between charged protein groups, and solid support
material (matrix).
Matrix has an ion load opposite to that of the protein to be
separated, and the affinity of the protein to the column is
achieved with ionic ties.
Proteins are separated from the column either by changing pH,
concentration of ion salts or ionic strength of the buffer solution.
Positively charged ion- exchange matrices are called anion-
exchange matrices, and adsorb negatively charged proteins.
While matrices bound with negatively charged groups are
known as cation-exchange matrices, and adsorb positively
charged proteins.
31.
32. REFERENCES
•Felgner P.L., et al. Lipofection: a highly efficient, lipid-mediated DNA-
transfection procedure. 1987, Proc Natl Acad Sci U S A,
10.1073/pnas.84.21.7413.
•Lee C.S., et al. Adenovirus-mediated gene delivery: Potential
applications for gene and cell-based therapies in the new era of
personalized medicine. Genes Dis, 2017,
10.1016/J.GENDIS.2017.04.001.
•Sakuma T., Barry M.A., Ikeda Y. Lentiviral vectors: basic to translational.
Biochem J, 2012, 10.1042/BJ20120146.
•Meissner P, Pick H, Kulangara A, et al. Transient gene expression:
recombinant protein production with suspension-adapted HEK293-EBNA
cells[J]. Biotechnol Bioeng, 2001, 75(2): 197-203.
•Cockett M I, Bebbington C R, Yarranton G T. High level expression of
tissue inhibitor of metalloproteinases in Chinese hamster ovary cells
using glutamine synthetase gene amplification[J]. Biotechnology (N Y),
