Scribd
Upload
45 views

Mammalian Expression System (Cell Line

The document summarizes protein production strategies in mammalian expression systems. It discusses both transient and permanent expression methods. For transient expression, the key steps are cloning the gene into a vector, transfecting cells, and harvesting the protein after 72 hours. For permanent expression, stable cell lines are generated by selecting transfected cells in media with antibiotics to integrate the gene into the genome. The document also describes inducible expression systems that control protein production using tetracycline.

Uploaded by

Aditi Verma
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
45 views

Mammalian Expression System (Cell Line

The document summarizes protein production strategies in mammalian expression systems. It discusses both transient and permanent expression methods. For transient expression, the key steps are cloning the gene into a vector, transfecting cells, and harvesting the protein after 72 hours. For permanent expression, stable cell lines are generated by selecting transfected cells in media with antibiotics to integrate the gene into the genome. The document also describes inducible expression systems that control protein production using tetracycline.

Uploaded by

Aditi Verma
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
You are on page 1/ 6

Lecture 27 Protein Production strategies in Expression System

(Part-IV)
Mammalian expression system: Similar to all other expression system, protein
production in mammalian cell system can be achieved with either from a vector present
as extrachromosomal DNA (transient ) or the sequence is integrated into the genome
through homologous recombination to establish the permanent cell line. The expression
from transient or permanent cell lines can be from a constituted or inducible promoter.
Irrespective of the expression mode in mammalian system, the different basic steps
needed to produce protein are as follows:

1. Cloning of foreign gene in mammalian expression vector.

2. Transfection of a cell line with recombinant construct.

3. Screening and selection of transfected cells.

4. Culturing of transfected cells.

5. Protein production.

Different Cell line for protein production: The most popular cell lines used for
mammalian expression system are given in Table 27.1. The cell lines are derived from
the different origin (organ of the body/cell type/animal) and consequently they are
preferred to produce foreign protein with similar origin.

Table 27.1: Popular Cell lines for protein production


S. No. Cell lines Origin
1 CV1 Kidney
2 COS-7 Fibroblast
3 3T3 Fibroblast
4 CHO Ovary
5 J774A.1 Macrophage
6 HeLa Cervical
7 BHK 21 Kidney
8 HEK 293 Kidney
Growth media for mammalian cell line: The growth media for different mammalian
cells is discussed earlier in a previous lecture.

Figure 27.1: Different mammalian cell lines used for protein production.

Protein Production in mammalian expression system:

Transient expression: The expression is high but for short time period. The cells
transfected with DNA express proein until 72h post-transfection. Transient expression
system is used to screen cDNA library, isolation of a particular cDNA clone expressing
surface antigen and to test the applicability of the recombinant construct going to use for
permanent expression. There are multiple steps required to transiently express a protein
in COS-7 cell line. Although we are giving the details for COS-7 but these procedures
can be applied to other cell lines with slight modifications (Figure 27.2). The steps are as
follows:

1. Clone the foreign DNA into the appropriate mammalian expression vector to obtain
recombinant DNA. Transfection efficiency is maximum for a supercoiled circular DNA.
Purify the recombinant DNA by a miniprep kit to prepare high quality supercoiled DNA.

2. Seed COS-7 cells in DMEM media supplemeted with 10% foetal bovine serum at 20%
confluence in 100mm dish.

3. Transfect the cells with recombinant DNA using a transfection reagent. [Several
transfection reagent and methods are discussed in a previous lecture].
If expressing secreted proteins-wash the transfected cells with PBS and add serum free
media. It allows to secrete the protein for next 72hr. Harvest the medium, remove dead
cells and debris by centrifugation and filter the media from 0.45µm syringe filter before
storage. Detect the presence of protein in media either by activity assay or by western
blotting.

If expressing cell surface or intracellular protein-Transfected cells are allowed to


express the protein for another 72 hrs. Remove media, wash the cells with PBS and detect
the presence of protein on cell surface or in cell lysate by activity assay or by western
blotting.

Figure 27.2: Different Steps in transient expression system.


Permanent expression: The permanent expression of a gene is possible, if it will be
integrated into the chromosomal DNA. The most crucial step to estabilish a permanent
expression system for a gene is the frequency of integration events rather than number of
DNA uptake. In simpler word, permanent transfection depends on recombination
frequency instead of transfection efficiency. Stable transformant are selected by a
selection marker (such as antibiotics or auxotrophic factor or negative selection with an
inhibitor) for a prolong period to ensure the integration of recombinant DNA into the
genome. The steps follow to generate a stable cell line expressing foreign protein is given
in Figure 27.3. The different steps are as follows-

Step 1-3 are indentical as discussed for the transient expression of a foreign gene.

4. Selection of trasnfected cells

4.1. Fourty eight hrs after transfection, split the transfected cells in a selection media
containing antibiotic and allowed to grow for another 4 days.

4.2. Gently wash the cells with PBS and observe the descrete colonies.

4.3. Delineate the boundry of each colony with a marker from the back side of the plate.

4.4. Remove the media and put cloning ring to each colony. Wash the colony with PBS
and add 100µl trypsin-EDTA to remove the colony.

4.5. wash the colony with PBS and transfer into one well of 24 well dish. Allow it to
grow and become 80% confluent.

4.6. Transfer these cells to the 6 well dish in the presence of selection media and allow it
to reach 80% confluency.

5. Take a small aliquot of the cell and test the expression of foreign protein with western
blotting. In addition, integration of gene into the genome can be checked by performing a
southern blotting with radioactive probe derived from the gene of interest.
Figure 27.3: Different Steps in permanent expression system.
Inducible expression system. Inducible expression system is useful for the expression of
a toxic protein or proteins with pleiotropic or non-specific effects. The tetracycline-
controlled inducible system is given in Figure 27.4. In this system, seven tandenly
arranged tet operator [(Tet-op)7] are placed upstream to the minimum CMV promoter and
transcriptional activator (tTA) gene. In another set, target gene is replaced with tTA gene.
In the presence of tetracycline, the binding of tTA is blocked to the tetracycline operator.
Consequently, it causes low level of expression of tTA and target gene. In the absence of
tetracycline, low level of tTA binds to the operator and drive the enhanced expression of
tTA which in-turn stimulates further amplification of initial signal. Transcription
activator (tTA) produced in the absence of tetracycline eventually stimulates the
expression of target gene.

Figure 27.4: Tet inducible system of mammalian expression system. Molecular events in the presence (A) or absence (B) of
tetracycline.

You might also like