11/27/2017 Protein Expression and Purification Core Facility - Cloning - Choice of Expression Systems - EMBL

Protein Expression and Purification

Core Facility
CLONING
CHOICE OF EXPRESSION SYSTEMS

After you have decided which protein or which domain(s) of a protein you would
like to clone and express, you have to think about which expression system you
would like to use. At present there are the following expression systems available:

Protein Expression Systems

Comparison of Expression Systems

Here we limit ourselves to the following three systems since they are most suited
for large-scale production of proteins:

— Escherichia coli: The expression of proteins in E. coli is the easiest, quickest and
cheapest method. There are many commercial and non-commercial expression
vectors available with different N- and C-terminal tags and many different strains
which are optimized for special applications (for local users: see strain database).

— Yeast: Yeast is an eukaryotic organism and has some advantages and disadvantages
over E. coli. One of the major advantages is that yeast cultures can be grown to very
high densities, which makes them especially useful for the production of isotope
labeled protein for NMR. The two most used yeast strains are Saccharomyces
cerevisiae and the methylotrophic yeast Pichia pastoris.

— Baculovirus infected insect cells: Insect cells are a higher eukaryotic system
than yeast and are able to carry out more complex post-translational modifications
than the other two systems (see Comparison of Expression Systems). They also
have the best machinery for the folding of mammalian proteins and, therefore, give
you the best chance of obtaining soluble protein when you want to express a protein
of mammalian origin. The disadvantages of insect cells are the higher costs and the
longer duration before you get protein (usually 2 weeks).

— Mammalian cells: Most labs use HEK (human embryonal kidney) or CHO
(Chinese hamster ovary) cell lines for preparative expression of more complex
proteins which also need proper post-translational modifications. Both cell lines

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can be used for both transient and stable cell line expression which is more time
consuming due to the generation of stable cell lines but offers higher productivity
and less variation if long-term production of a target protein is required. While
these cells have usually a high capacity of producing secreted protein (up to 10s or
100s of mg per Liter, often several grams per Liter in cell lines for commercial
proteins), their expression levels for intracellular proteins is usually much lower.

To determine which system is the best choice, ask yourself the following questions:

1. What type of protein do I want to express?

When you would like to express a protein of prokaryotic origin, the obvious choice
is to use E. coli. The method is quick and cheap and the organism has all the
machinery necessary for folding and post-translational modifications.

In case the protein is from an eukaryotic source, the method of choice will depend
on more factors (see below).

2. Do I get soluble protein when I express in E. coli ?

Also for the expression of eukaryotic proteins the first method of choice is normally
E. coli for the above mentioned reasons. However, many eukaryotic proteins don't
fold properly in E. coli and form insoluble aggregates (inclusion bodies). Sometimes
it is possible to resolubilize the protein from the inclusion bodies or improve the
solubility by expressing the protein at a lower temperature. Also expression of your
target protein as a fusion protein with a highly soluble partner such as glutathione-
S-transferase (GST), maltose binding protein (MBP), or DsbA can improve its
solubility. Often, however, it is better to change to an eukaryotic expression system
because it is better equipped to fold proteins from an eukaryotic source. Thus,
instead of trying out 10 different E. coli constructs, it is better to switch
expression system.

3. Does my protein need post-translational modifications for

structure/activity?

Many proteins need to be modified following translation in order to become active

and/or adapt the proper structure. The simplest of these modifications is the
removal of the N-terminal methionine residue, which can occur in all organisms.
More complex modifications, like N- and O-glycosylation, phosphorylation, are

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exclusively carried out by eukaryotic cells. Keep in mind that not all eukaryotic
cells carry out the same modifications. Check table 1 to find out which expression
system carries out the post-translational modification(s) you are looking for.

4. What is the codon usage in my protein?

Not all of the 61 mRNA codons are used equally. The so-called major codons are
those that occur in highly expressed genes, whereas the minor or rare codons tend
to be in genes expressed at a low level. Which of the 61 codons are the rare ones
depends strongly on the organism. The codon usage per organism can be found in
the Codon Usage Database. For more information on the low usage codons per
organisms see table 2 and table 3.

Usually, the frequency of the codon usage reflects the abundance of their cognate
tRNAs. Therefore, when the codon usage of the protein you would like to express
differs significantly from the average codon usage of the expression host, this could
cause problems during expression. The following problems are often encountered:

— Interrupted translation, which leads to a variety of truncated protein products.

— Frame shifting.

— Misincorporation of amino acids. For instance, lysine for arginine as a result of

the AGA codon. This can be detected by mass spectroscopy since it causes a
decrease in the molecular mass of the protein of 28 Da.

— Inhibition of protein synthesis and cell growth.

As a consequence, the observed levels of expression are often low or there will be
no expression at all. Especially in cases were rare codons are present at the 5'-end
of the mRNA or where consecutive rare codons are found expression levels are low
and truncated protein products are found.

To increase the expression levels of proteins containing rare codons in E. coli, two
main methods are available:

— Site-directed mutagenesis to replace the rare codons by more commonly used

codons for the same residue; e.g. the rare argenines codons AGA and AGG by the E.
coli preferred CGC codon.

— Co-expression of the genes which encode rare tRNAs. There are several
commercial E. coli strains available that encode for a number of the rare codon

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genes:

BL21 (DE3) CodonPlus-RIL AGG/AGA (arginine), AUA Stratagene

(isoleucine) and CUA
(leucine)

BL21 (DE3) CodonPlus-RP AGG/AGA (arginine) and Stratagene

CCC (proline)

Rosetta or Rosetta (DE3) AGG/AGA (arginine), CGG Novagen

(arginine), AUA (isoleucine)
CUA (leucine), CCC
(proline), and GGA (glycine)

Often you will obtain a mixture of full-length protein and truncated species.
Providing the protein with a C-terminal tag ( e.g. His6-tag) will help you to purify
only the full-length protein using affinity chromatography.

When both above-mentioned methods fail to increase expression levels, it is time to

change expression system and try to express your protein in yeast or insect cells. In
cases where the protein contains many rare E. coli codons it is probably better to
immediately start with an eukaryotic system.

References

Protein Expression. A practical approach (Higgins. S.J. & Hames, B.D., eds), Oxford
University Press, 1999.

Kane, J.F. (1995) Effects of rare codon clusters on high-level expression of

heterologous proteins in Escherichia coli. Current Opinions Biotechnol. 6, 494-500.
Zhang, S., Zubay, G. & Goldman, E. (1991) Low-usage codons in Escherichia coli,
yeast, fruit fly and primates. Gene 105 , 61-72.

Novy, R., Drott, D., Yaeger, K. & Mierenhof, R. (2001) Overcoming the codon bias of
E. coli for enhanced protein expression. inNovations 12 , 1-3.

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