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Dual
Media Set™
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Reliable method for high level recombinant protein expression
in E. coli. |
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Simple
procedure: inoculate bacteria into EB™ medium
for cell expansion, add OB™ medium for protein
over-expression. |
| • |
Eliminates
the need to monitor culture density and to optimize timing
of inducer addition. |
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Synchronizes
cultures expressing diverse recombinant proteins.
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| DESCRIPTION
Although
recombinant protein expression in E. coli has become
all but routine, high level protein expression or overexpression
is not always consistent and repeatable for every protein.
Our research at Zymo Research Corporation has shown that
high level protein expression can be achieved consistently
when two processes, cell expansion and protein expression,
are kept completely separate.
The Dual Media Set™, different from
commonly used protein expression procedures using Luria
Broth (LB) or other specially prepared medium, contains
two media, EXPANSION BROTH™ (EB™)
and OVEREXPRESSION BROTH™ (OB™).
For cell expansion, E. coli cells are grown in
EB™, while the production of the
recombinant protein is almost completely repressed. For
high level protein expression, the expanded cell culture
is simply added into OB™ media (see
Figure 1). By using the Dual Media Set™ system,
protein overexpression can be reliably controlled for many
proteins, as demonstrated in Figure 2.
In some circumstances, such as when expressing toxic proteins,
or when the expressed protein is poorly soluble, over-expression
is counterproductive. In such cases, limited protein production
can be initiated by adding the specific inducer (IPTG for
lac-based promoters) to cells growing in EB™ (see
Figure 1 and Protocol II).
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ORDERING INFORMATION:
| Products |
Protocol |
Cat
No. |
Size |
Price |
|
Dual
Media Set™
(contains 100 ml EB™ and 500 ml OB™)
|
|
M3011 |
1
Set |
$38.00 |
|
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Components
Available Separately |
|
Expansion
Broth™ (EB™) |
M3012-100 |
100
ml |
$12.00 |
|
| M3012-500 |
500
ml |
$28.00 |
|
|
Overexpression
Broth™ (OB™) |
M3013-100 |
100
ml |
$12.00 |
|
| M3013-500 |
500
ml |
$28.00 |
|
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What underlying mechanism ensures
that the Dual Media Set™ works so reliably for protein
overexpression?
Our research has found that the
use of the common Luria Broth (LB) medium produces relatively
high background levels of recombinant protein expression even
without the inducer (such as IPTG). The expression often does
not increase enough after addition of the inducer. We have
concluded that although LB is an excellent medium for normal
E. coli manipulation, it is not a good medium for
repression of protein expression during cell expansion, nor
is it ideal for protein expression when inducer is added.
EB™ and OB™ were designed to overcome these problems.
EB™ was designed to repress recombinant protein expression
by regulating the levels of cyclic AMP (cAMP) and cAMP receptor
protein (CRP) during cell expansion, so that the cells can
be expanded without pressure of undesired foreign protein
expression and unintentional selection of mutated clones to
overgrow original inoculates. When the cells are expanded,
OB™ medium is added for protein expression. During this
process, further cell growth is not required and selection
of new mutants no longer occurs because cell replication is
very limited at this stage. The carefully formulated composition
of OB™ works similarly by regulating the levels of cAMP
and CRP but with an opposite effect than EB™. Therefore,
addition of IPTG is not needed for strong promoters such as
T7.
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What
are the ingredients of EB™ and OB™ ?
Our growth media contain only
standard commonly used ingredients, salts, and carbohydrates.
The final composition, and the ratio of individual components,
has been carefully optimized to result in above described
metabolic effects.
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Can I replace EB™ or OB™
with my home made media?
EB™ can be replaced with
LB when the expressed protein is stable and does not interfere
with cell growth. OB™ cannot be replaced.
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Can I use OB™ without the
initial cell expansion in EB™ ?
The simplified protocol can be
used, but only for most stable and easily expressed proteins.
You can simply start at step 2. in Protocol I. This will result
in strong protein overexpression, with the risk of introducing
unwanted mutations or other unwanted effects.
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I have always used IPTG to induce
protein expression in the T7 system, why not now? Will I get
less protein if I don't use IPTG in such case?
The T7 promoter is too strong
when induced with IPTG. The metabolic effects caused by growth
in OB™ are also strong. This is a powerful combination,
always resulting in too rapid protein expression with negative
effects on cell viability. This situation results in lower
yields of recombinant protein and may lead to additional plasmid/protein
instability.
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Using the EB/OB system, what volume
of bacterial culture do I need to get a satisfactory amount
of protein for my experiments? Using the traditional LB/IPTG
system, I had to grow 1 liter of culture.
Protein yields vary considerably depending on strain, protein,
and culture conditions. However, a simple preliminary calculation
is possible: Let's take the example of a 1 liter culture.
Normally, an LB culture reaches a maximum optical density
at 600 nm (OD600) of no more than 4.0. An OB culture
grows up to more than OD600 = 20. That means approximately
5x more cells can be obtained from the OB culture.
The second factor is protein expression - this tends to be
variable. A conservative estimate assumes that each cell will
produce 2x as much recombinant protein using the EB/OB system
(and you will likely get much more than that). Combining these
two factors gives us 2 x 5 = 10, which means that you only
need 10 times less culture volume when using the Dual Media™
Set. In our practical example, you will start with 25 ml of
EB culture and then add 75 ml of OB, bringing the final volume
to 100 ml.
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| 7. |
What do these media have in common
with Novagen's Overnight Express™ Autoinduction System
(Novagen - EMD Biosciences, Inc., Madison, WI)?
Zymo's Dual Media Set™ (EB™
and OB™) completely uncouples cell expansion and protein
expression (see above), enabling better control over the entire
process in order to avoid mutations or low expression of toxic
proteins. Novagen's media, however, does not allow complete
separation of cell expansion and protein expression (see above),
which may result in lower protein yields, mutations, and other
unwanted effects. While Novagen's media are used only for the
T7 system, Zymo's Dual Media Set™ works well with the
T7 system as well as with all lac based promoters and
a number of other catabolic promoters.
The metabolic effects brought about by growth in Novagen's media
may be due to similar principles and may yield results similar
to those obtained by growth in Zymo's Dual Media Set™
- i.e. a strong protein overexpression. Both systems offer IPTG-free
induction without the need to monitor cell density, thus eliminating
the most laborious part of the process.
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REFERENCES:
Kelley KC, Huestis KJ, Austen DA, Sanderson CT, Donoghue MA,
Stickel SK, Kawasaki ES, Osburne MS. Regulation of sCD4-183
gene expression from phage-T7-based vectors in Escherichia
coli. Gene. 1995 Apr 14;156(1):33-6.
Grossman
TH, Kawasaki ES, Punreddy SR, Osburne MS. Spontaneous cAMP-dependent
derepression of gene expression in stationary phase plays
a role in recombinant expression instability. Gene. 1998
Mar 16;209(1-2):95-103.
Botsford
JL, Harman JG. Cyclic AMP in prokaryotes. Microbiol Rev.
1992 Mar;56(1):100-22.
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| Click
image to enlarge |
Figure
1. SDS-PAGE electrophoresis of cell proteins after growth
in Dual Media Set™.
M – protein
markers;
1-5, West Nile virus protein E (54
kDa): 1, repressed expression in EB™, 2-5, overexpression
in OB™ for 5, 10, 18, and 24 hours, respectively, after inoculation
with uninduced EB™ culture;
6-10, C-terminal domain of West Nile
virus protein E (32 kDa): 6, repressed expression in EB™,
7-10, overexpression in OB™ for 5, 10, 18, and 24 hours, respectively,
after inoculation with uninduced EB™ culture;
11-15, Methyl-binding domain of MeCP2
(18 kDa): 11, repressed expression in EB™, 12-15, overexpression
in OB™ for 5, 10, 18, and 24 hours, respectively, after inoculation
with uninduced EB™ culture; |
 |
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Click
image to enlarge |
| Figure
2: Controlled overexpression of ß-galactosidase: cell culture
is first expanded in EB™, where only background levels of
the T7-lac promoter-controlled product are produced (1). Moderate
amounts of the enzyme are produced by overnight incubation in EB™
with IPTG (2), highest amounts of protein are produced in OB™
(3). |
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