E coli advantages

Recombinant Protein Expression in E.coli

INQUIRY

Background
Recombinant proteins are widely used in biological and biomedical research[1],
and recombinant protein expression has become a commonly used tool.
Nevertheless, there are still many aspects to consider before starting an
expression project. For example, which expression system is chosen for
obtaining the protein of interest? Should it be expressed in bacteria, in yeast, in
insect cells or in mammalian cells? What is the best expression vector to be
used? As to the bacterial expression system, which strain should be chosen?
Is there any differences in the expression condition between the full-length
protein and a protein fragment? Which affinity tag should be used to facilitate
both protein expression and subsequent purification steps? How to work out
an appropriate purification strategy? Since protein property can vary a lot, all
conditions need to be carefully chosen to fit the project requirements.

Profacgen is equipped with experienced scientific team and well-developed

recombinant protein expression systems to serve the biopharmacetutical and
life science field fully customized recombinant protein production services. Our
team can help customers work out a best fit expression plan and purification
strategy for the target protein with its intended application. Over the past 10
years, our Profacgen team has targeted and purified thousands of proteins
from the prokaryotic systems and eukaryotic system with both structural
integrity and functional activity.

Of all the expression hosts, E.coli is usually the first choice when applicable
because of its ease of manipulation and low cost.

Advantages of E.coli expression system

Profacgen has extensive experience in optimizing E.coli host strain and

expression vectors for more efficient recombinant protein production. Various
proteins, from prokaryotic proteins to eukaryotic proteins, from enzymes to
transcription factors, from full-length to truncated domains, from monomeric
protein to large protein complexes can be produced in our specialized E.coli
expression system. For those proteins that do not express in soluble form due
to misfolding in the E.coli system, we also have developed strategies to extract
them from the inclusion bodies.
For high-level protein production, BL21(DE3) is a basic and most widely used
E.coli strain. It has advantages of being deficient in both lon and ompT
proteases and is compatible with the T7 lacO promoter system [2]. Our
scientists have also developed several BL21 derivatives carrying additional
helper plasmid to overcome the bad effects of codon bias in protein
expression.

Protocol for Recombinant protein expression in E.coli

1. Small-scale expression evaluation & expression optimization

Our service allows you to evaluate your target protein expression in your
chosen expression system; identify the construct and conditions that gives you
the most robust soluble expression of your target protein. We can work with
you to design the expression trial according to your exact requirements.
Validation of soluble protein expression in a chosen E.coli strain generally
takes 1-2 weeks.

Small-scale expression evaluation & expression optimization

2. Standard Procedure:

a. Molecular cloning:

The gene of interest is inserted into a chosen expression vector. Usually the
target protein is fused to a protein tag to facilitate expression and purification
(i.e., 6*Histidine). The protein tag can be inserted to the N-terminus or
C-terminus of the target gene for fusion protein expression [3]. Choice and
position of the protein tag usually depends on the property of the target protein,
expression condition and the customer’s application. A protease cleavage site
can be inserted between the target protein and the protein tag, which will allow
removal of the protein tag after purification. In addition, the expression vector
also contains antibiotic resistance markers for clone selection [4].

Sub-cloning Word-flow

b. Transformation of the expression vector into chemically competent cells.

Competent cells are cells that can readily take up foreign DNA that, in this case,
contain the target protein for expression. Below is a standard protocol for most
expression vectors and cell lines [5].

i. Add the target vector into thawed competent cells.

ii. Incubate the cells on ice, then treat with heat-shock and ice-shock.
iii. Add liquid medium into treated competent cells for recovery.
iv. Plate the transformed cells onto agar plates containing corresponding
antibiotic for clone selection.
c. Expression tests.

In order to identify the optimal conditions for growth and expression of the
target protein, we perform complicated expression trials of temperature,
induction time and expression time.

i. Transform the chosen E.coli strain competent cells for expression with the
constructed expression vector.
ii. Select the appropriate colony by antibiotic screening.
iii. Incubate the correct colony harboring the recombinant plasmid in LB until
OD600 reached to a certain value.
iv. Divide the culture, equilibrate at different chosen temperatures, and induce
expression by addition of isopropyl-β-D-thiogalactoside (IPTG).
v. Continue cell growth and take samples at different time points after
induction.
vi. The remaining cultures were allowed to continue growing and eventually
harvested.
vii. All the samples were then lysed by sonication and then centrifuged to
separate the supernatant and pellet for sodium-dodecyl sulphate
polyacrylamide gel electrophoresis (SDS-PAGE) analysis.
viii. Subject the re-suspended cells to a single freeze-thaw cycle prior to
DNase digestion.
ix. Centrifuge the lysed cell to remove the cell debris and filter the supernatant
through a filter membrane.
Please note that parameters such as OD600 value prior to IPTG induction,
IPTG concentration, expression temperature and harvest time point can be
adjusted to the expression status of the recombinant protein.

d. Protein purification

Preparation of the bacterial lysate can be critical for the subsequentprotein

purification. The right cell lysis condition can maximize recombinant protein
extraction, minimize unwanted proteolysis and sample contamination with
genomic DNA and protein oxidation [1].

i. Cell lysis. Add lysozyme to the lysis buffer during mechanical lysis by
sonication or lysis by freeze-thaw procedure. PH and salt concentration of the
lysis buffer needs to be optimized to enhance protein solubility and stability
according to the physiochemical property (solubility and pI, etc.) of the target
protein. Protease inhibitors and reducing agent such as Tris (2-carboxyethyl)
phosphine hydrochloride (TCEP) are used to protect target protein from
complicated cell lysate mixture.
ii. Protein purification by chromatography. One huge advantage of
recombinant protein is that purification is greatly simplified by the addition of
protein purification tags. Specific binding of certain metal or antibody of the
protein tags can therefore be used in affinity chromatography to achieve strong
and specific binding of the target protein.
Protein purification by chromatography

Profacgen has implemented various protein purification protocols on

automated chromatography systems to achieve reliable and consistent results.
SDS-PAGE analysis is used to determine the target protein yield from the
previous small-scale expression trials, which also determines the amount of
cell lysate to be loaded on the column. SDS-PAGE is also used to reveal the
purification efficiency and purity of protein after each purification step.
Click here to contact us for more technical information.

References:
[1]Structural Genomics C, China Structural Genomics C, Northeast Structural
Genomics C, et al. Protein production and purification[J]. Nat Methods, 2008,
5(2): 135-46.
[2]Studier F W, Rosenberg A H, Dunn J J, et al. Use of T7 RNA polymerase to
direct expression of cloned genes[J]. Methods Enzymol, 1990, 185: 60-89.
[3](2) M-Q D P a R H G B. Mutation of the Catalytic Cysteine in Anopheles
gambiae Transglutaminase 3 (AgTG3) Abolishes Plugin Crosslinking Activity
without Disrupting Protein Folding Properties[J]. Journal of Emerging
Investigators, 2014.
[4]Davies H A. Expression and characterisation of cardiovascular amyloid
proteins.[D]. University of Liverpool, 2013: 288.
[5]Sambrook J, Fritsch, E. F., Maniatis, T. Molecular Cloning - A laboratory
handbook.[M]. In: NOLAN, C. (ed.) 2 ed.: Cold Spring Harbour Laboratory
Press., 1989.