PROTEIN EXPRESSION AND ELECTROPHORETIC TECHNIQUE IN

MOLECULAR BIOLOGY

PROTEIN EXPRESSION

The central dogma in molecular biology is DNA→RNA→Protein. To synthesize a particular

protein DNA must first be transcribed into messenger RNA (mRNA). mRNA can then be
translated at the ribosome into polypeptide chains that make up the primary structure of
proteins. Most proteins are then modified via an array of post-translational modifications
including protein folding, formation of disulfide bridges, glycosylation and acetylation to
create functional, stable proteins. Protein expression refers to the second step of this process:
the synthesis of proteins from mRNA and the addition of post-translational modifications

Researchers use various techniques to control protein expression for experimental,

biotechnological, and medical applications. Researches can visualize proteins in vivo by
tagging them with fluorescent proteins to study localization or purify proteins to study their
structure, interactions and functions. Proteins can also be purified for use in molecular
biology research (eg. polymerases and other enzymes might be purified and used to
manipulate DNA), or in medicine (e.g. insulin).

Proteins, unlike DNA which can be relatively easily synthesized, must be produced using
complex mixtures derived from cells or using live cells. There are several types of expression
systems used for protein production and purification. These include mammalian, insect,
bacterial, plant, yeast and cell free expression systems.
Overall the general strategy for protein expression consists of transfecting cells with your
DNA template of choice and allowing these cells to transcribe, translate and modify your
protein of interest. Modified proteins can then be extracted from lysed cells through the use
of protein tags and separated from contaminants using a variety of purification methods.
Deciding which expression system to use depends on several factors:

1. The protein you are trying to express

2. How much protein you need
3. Your plans for downstream applications

SUMMARY OF SOME COMMON EXPRESSION SYSTEMS INCLUDING THEIR

ADVANTAGES

MAMMALIAN EXPRESSION SYSTEMS

Mammalian cells are an ideal system for the expression of mammalian proteins that require
multiple post-translation modifications for proper protein function. Most DNA constructs
designed for mammalian expression utilize viral promoters (SV40, CMV, and RSV) for
robust expression post-transfection. Mammalian systems can express proteins both transiently
and through stable cell lines. Both methods produce high protein yields if transfection is
successful.
Some mammalian systems also allow for control over when a protein is expressed through
the use of constitutive and inducible promoters. Inducible promoters are extremely useful if a
desired protein product is toxic to cells at high concentrations. Despite their advantages,
mammalian expressions systems do require demanding cell culture conditions compared to
other systems.

INSECT EXPRESSION SYSTEMS

Insect cells can also be used to produce complex eukaryotic proteins with the correct post-
translational modifications.

There are two types of insect expression systems; baculovirus infected and non-lytic insect
cells.

Baculovirus expression systems are very powerful for high level recombinant protein
expression. These systems enable high expression of very complex, glycosylated proteins that
cannot be produced in E. coli or yeast cells. The only problem with baculovirus systems is
that the infected host cell is eventually lysed. Cell lysis halts protein production, but there are
non-lytic insect cell expression systems (sf9, Sf21, Hi-5 cells) that allow for continuous
expression of genes integrated into the insect cell genome. Both of these types of insect
expression systems can be scaled up for production of large amounts of protein. Some
drawbacks of insect cell expression systems include that virus production can be quite time
consuming and that insect cells require demanding culture conditions similar to mammalian
expressionsystems

BACTERIAL EXPRESSION SYSTEMS

When one wants to produce vast quantities of protein rapidly and cheaply, a bacterial host
cell is almost always the answer. E. coli is definitely one of the most popular hosts for protein
expression with several strains that are specialized for protein expression. Protein expression
in bacteria is quite simple; DNA coding for your protein of interest is inserted into a plasmid
expression vector that is then transformed into a bacterial cell. Transformed cells propagate,
are induced to produce your protein of interest, and then lysed. Protein can then be purified
from the cellular debris.
There are several popular DNA vectors that can be used to produce large amounts of protein
in bacterial cells: the pET, pRSET, Gateway pDEST, and pBAD vectors for example. Protein
expression from each of these vectors is controlled by a different promoter resulting in
different levels of expression from each vector; lower expression may be required if your
protein is toxic to E. coli. Of all the vectors, pET, under the control of the T7 lac promoter
and induced by lactose, provides the highest level of protein expression.

Despite their ease of use, it is important to note that bacteria usually cannot produce
functional multi-domain mammalian proteins as bacterial cells are not equipped to add
appropriate post-translational modifications. In addition many proteins produced by bacteria
become insoluble, forming inclusion bodies that are difficult to extract without harsh reagents
and patience.

PLANT EXPRESSION SYSTEMS

Plants provide a cheap and low-tech means of mass expression of recombinant proteins.
Many cells from various types of plants such as maize, tobacco, rice, sugarcane, and even
tubers of potatoes have been used for protein expression.

Plant systems share many of the same features and processing requirements as mammalian
cell expression systems, including the majority of complex post-translational modifications.
Extraction and purification of recombinant proteins from plants however can be expensive
and time consuming as plant tissues themselves are biochemically complex.

To circumvent these issues, scientists have taken advantage of the natural secretion of
biochemicals and proteins through plant roots. Tagging recombinant proteins with a naturally
secreted plant peptide allows for easier access and purification of a desired protein. Despite
being a rather nascent technology, plant cells have been used to express a wide range of
proteins including antibodies and pharmaceuticals, specifically interleukins.

Yeast expression systems

Yeast are a great expression system to generate large quantities of recombinant eukaryotic
proteins. Although many species of yeast can be used for protein expression, S. cerevisiae, is
the most reliable and frequently used species due to its use as a model organism in genetics
and biochemistry.
 When using S. cerevisiae, researchers often place recombinant proteins under the control of
the galactose inducible promoter (GAL). Other commonly used promoters include the
phosphate and copper inducible PHO5 and CUP1 promoters respectively. Yeast cells are
grown in well-defined media and can be easily adapted to fermentation allowing for large-
scale, stable production of proteins.

In general yeast expression systems are easier and cheaper to work with than mammalian
cells, and are often capable of modifying complex proteins unlike bacterial systems. Yeast
cells, however, have a slower growth rate than bacterial cells and growing conditions often
need to be optimized. Yeast cells are also known for hyperglycosylating proteins which may
be an issue depending on your protein of choice.

Cell free expression systems

In cell-free expression systems, proteins are assembled in vitro using purified components of
the transcription and translation machinery. These include ribosomes, RNA polymerase,
tRNAs, ribonucleotides and amino acids. Cell-free expression systems are ideal for fast
assembly of more than one protein in one reaction. A major advantage of these systems
system is their ability to assemble proteins with labelled or modified amino acids that are
useful in different downstream applications. Cell-free expression systems however, are
expensive and very technically challenging to use.

ELECTROPHORETIC TECHNIQUE IN MOLECULAR BIOLOGY

Gel electrophoresis

 Gel electrophoresis is a technique used to separate DNA fragments according to their size.

 DNA samples are loaded into wells (indentations) at one end of a gel, and an electric current
is applied to pull them through the gel.

 DNA fragments are negatively charged, so they move towards the positive electrode. Because
all DNA fragments have the same amount of charge per mass, small fragments move through
the gel faster than large ones.

 When a gel is stained with a DNA-binding dye, the DNA fragments can be seen as bands,
each representing a group of same-sized DNA fragments.
Introduction

Suppose you have just done a PCR reaction, making many copies of a target DNA
region. Or perhaps you’ve done some DNA cloning, trying to "paste" a gene into a
circular DNA plasmid.

Now, you want to check and see whether your PCR worked, or whether your plasmid
has the right gene in it. What technique can you use to visualize (directly observe) the
fragments of DNA?

Gel electrophoresis

Gel electrophoresis is a technique used to separate DNA fragments (or other macromolecules,
such as RNA and proteins) based on their size and charge. Electrophoresis involves running a
current through a gel containing the molecules of interest. Based on their size and charge, the
molecules will travel through the gel in different directions or at different speeds, allowing
them to be separated from one another.

All DNA molecules have the same amount of charge per mass. Because of this, gel
electrophoresis of DNA fragments separates them based on size only. Using electrophoresis,
we can see how many different DNA fragments are present in a sample and how large they
are relative to one another. We can also determine the absolute size of a piece of DNA by
examining it next to a standard "yardstick" made up of DNA fragments of known sizes.

What is a gel?

As the name suggests, gel electrophoresis involves a gel: a slab of Jello-like material. Gels
for DNA separation are often made out of a polysaccharide called agarose, which comes as
dry, powdered flakes. When the agarose is heated in a buffer (water with some salts in it) and
allowed to cool, it will form a solid, slightly squishy gel. At the molecular level, the gel is a
matrix of agarose molecules that are held together by hydrogen bonds and form tiny pores.

At one end, the gel has pocket-like indentations called wells, which are where the DNA
samples will be placed:
A sketch of a gel electrophoresis apparatus(There is a power source with 2 lines extending
from the power source to the apparatus. One line goes to the top of the apparatus and is
attached and labeled with a negative symbol. The other line goes to the bottom of the
apparatus and is attached and labeled with a positive symbol. At the top of the apparatus there
are 4 small boxes with arrows pointing at each box and labeled wells. The middle of the tray
in the apparatus has an arrow pointing to it and is labeled gel)

Before the DNA samples are added, the gel must be placed in a gel box. One end of the box
is hooked to a positive electrode, while the other end is hooked to a negative electrode. The
main body of the box, where the gel is placed, is filled with a salt-containing buffer solution
that can conduct current. The buffer fills the gel box to a level where it just barely covers the
gel.

The end of the gel with the wells is positioned towards the negative electrode. The end
without wells (towards which the DNA fragments will migrate) is positioned towards the
positive electrode.

How do DNA fragments move through the gel?

Once the gel is in the box, each of the DNA samples we want to examine (for instance, each
PCR reaction or each restriction-digested plasmid) is carefully transferred into one of the
wells. One well is reserved for a DNA ladder, a standard reference that contains DNA
fragments of known lengths. Commercial DNA ladders come in different size ranges, so we
would want to pick one with good "coverage" of the size range of our expected fragments.
Next, the power to the gel box is turned on, and current begins to flow through the gel. The
DNA molecules have a negative charge because of the phosphate groups in their sugar-
phosphate backbone, so they start moving through the matrix of the gel towards the positive
pole. When the power is turned on and current is passing through the gel, the gel is said to
be running.

DNA samples are loaded into wells at negative electrode end of gel.

Power is turned on and DNA fragments migrate through gel (towards the positive electrode).

After the gel has run, the fragments are separated by size. The largest fragments are near the
top of the gel (negative electrode, where they began), and the smallest fragments are near the
bottom (positive electrode).
As the gel runs, shorter pieces of DNA will travel through the pores of the gel matrix faster
than longer ones. After the gel has run for awhile, the shortest pieces of DNA will be close to
the positive end of the gel, while the longest pieces of DNA will remain near the wells.

Very short pieces of DNA may have run right off the end of the gel if we left it on for too
long.

Visualizing the DNA fragments

Once the fragments have been separated, we can examine the gel and see what sizes of bands
are found on it. When a gel is stained with a DNA-binding dye and placed under UV light,
the DNA fragments will glow, allowing us to see the DNA present at different locations
along the length of the gel.

An x-ray image of a gel electrophoresis blot(The image has 2 columns on a black background
and to the left of the columns are the labels from top to bottom of 5000 bp, 1500 bp and
500bp. The first column of the image is labeled ladder, and there are multiple white lines
visible from the top to the bottom of the column against the black background. The second
row of the image is labeled PCR reaction and there is one thick white band located near the
500 bp label.
The bp next to each number in the ladder indicates how many base pairs long the DNA
fragment is.

A well-defined “line” of DNA on a gel is called a band. Each band contains a large number
of DNA fragments of the same size that have all travelled as a group to the same position. A
single DNA fragment (or even a small group of DNA fragments) would not be visible by
itself on a gel.