Expression of Soluble Proteins with NativeFolderTM

NativeFolderTM
Bacterial Culture Medium for the Expression of Soluble Proteins

o  Despite the development of Yeast, Insect and Mammalian cells expression
systems, E. coli remains the recombinant expression system the most
widely used by researchers worldwide.
o  E. coli accounts for 73% of all host cells used in recombinant protein
expression.(1)
o  E. coli’s expression system accounts for more than 3.5X of all other
recombinant protein expression systems…combined.(1)
o  E. coli expression systems are:
o  Easy (little to no training or equipment needed)
o  Inexpensive
o  Fast
o  Scalable
(1) Front. Microbiol., 05 March 2014 | http://dx.doi.org/10.3389/fmicb.2014.00085
E. coli remains the Host of Choice for Recombinant
Protein Expression
© Molecular Depot LLC. Address: 7915 Silverton Avenue, Suite 313, San Diego, CA 92126.
Web: https://moleculardepot.com. Email: info@moleculardepot.com. Phone: 858-900-3210

1)  Low or zero expression levels because of codon usage issues, toxicity, or
cytoplasmic degradation.
2)  Mis-folding, aggregation or low solubility leading to the formation of
inclusion bodies.
3)  Production of inactive proteins due to mis-folding, mis-placement of
disulfide bonds, etc.
Protein Expression in E. coli can, Sometimes, Go Wrong

o  NativeFolderTM is a bacterial culture medium specially formulated to
promote the folding and the expression of soluble recombinant proteins in
E. coli.
o  NativeFolderTM contains proprietary compounds that prevent protein
aggregation and misfolding during protein expression.
o  NativeFolderTM spares researchers precious time and effort experimenting
with the renaturation of inclusion bodies (which rarely works).
o  NativeFolderTM promotes the expression of correctly folded, soluble, target
proteins.
o  Recombinant proteins of up to 110 kDa were successfully expressed as
soluble fractions using NativeFolderTM.
NativeFolderTM Bacterial Culture Medium

LB NF LB NF LB NF
Wild-type
Comparative Expression of a 27 kDa Protein in LB and NativeFolderTM
Protein 1 is a human enzyme with a MW of 27 kDa. Wild-type and two mutant versions of Protein 1 were cloned
and expressed in E. coli strain BL21(DE3), using either the standard Luria Broth culture medium (LB) or the
NativeFolderTM bacterial culture medium (NF). Upon expression and cell lysis, the soluble fraction was recovered
by centrifugation and analyzed by SDS-PAGE. Results show that, unlike LB, NativeFolderTM is very effective at
expressing Protein 1 (wild-type and mutant versions) as soluble proteins. M: Mark12TM Protein Ladder (kDa).
200
116.3
97.4
66.3
55.4
36.5
31.0
21.5
14.4
6.0
Mutant 1 Mutant 2
M
Molecular Depot LLC. Address: 7915 Silverton Avenue, Suite 313, San Diego, CA 92126.
Web: https://moleculardepot.com. Email: info@moleculardepot.com. Phone: 858-900-3210.
Protein 1
(27 kDa)

T S P T S P
Low Temperature
(15°C)
High Temperature
(37°C)
Expression of a 27 kDa Protein Using NativeFolderTM at Different Temperatures
Protein 1 is a 27 kDa protein that exclusively forms inclusion bodies when expressed in standard culture
media. When expressed using NativeFolderTM, at low temperature, Protein 1 is mostly found in the soluble
fraction (S, > 90%). Interestingly, even when bacteria are grown at higher temperature 37°C, protein 1 is still
found in the soluble fraction, albeit at a lower percentage. M: Mark12TM Protein Ladder (kDa). T: Crude Extract, S:
Soluble fraction (cell-free extract), P: Pellet.
200
116.3
97.4
66.3
55.4
36.5
31.0
21.5
14.4
6.0
Protein 1
(27 kDa)

M LB NF
Comparative Expression of an 83 kDa Protein in LB and NativeFolderTM
Protein 2 (83 kDa) was cloned and expressed in E. coli strain BL21(DE3), using either the standard Luria Broth
culture medium (LB) or the NativeFolderTM bacterial culture medium (NF). Upon expression and cell lysis, the
soluble fraction was recovered by centrifugation and analyzed by SDS-PAGE. Results show that, unlike LB,
NativeFolderTM is very effective at expressing Protein 2 as a soluble protein. M: Protein Ladder (kDa)
Protein 2
(83 kDa)

Purification of an 83 kDa Protein after Expression in NativeFolderTM
Protein 2 (83 kDa) was expressed in the NativeFolderTM bacterial culture medium. Bacterial pellet was
resuspended and lysed in the NativeLyserTM buffer and the cell-free extract purified on a Ni-NTA affinity column
followed by a Q anion exchange column in tandem with an SP cation exchange column.
Protein 2
(83 kDa)
Ni-NTA Q-SP tandem 1
Q-SP tandem 2
Final Product

M LB NF
Comparative Expression of a 109.3 kDa Protein in LB and NativeFolderTM
Protein 3 (109.3 kDa) was cloned and expressed in E. coli strain BL21(DE3), using either the standard Luria Broth
culture medium (LB) or the NativeFolderTM bacterial culture medium (NF). Upon expression and cell lysis, the
soluble fraction was recovered by centrifugation and analyzed by SDS-PAGE. Results show that, unlike LB,
NativeFolderTM is very effective at expressing Protein 3 as a soluble protein.
M: Mark12TM Protein Ladder (kDa)
200
116.3
97.4
66.3
55.4
36.5
31.0
21.5
14.4
6.0
Protein 3 (109.3 kDa)

M CFE FT W E1 E2 E3 E4 E5 E6
Purification of a 109.3 kDa Protein after Expression in NativeFolderTM
His-tagged Protein 3 (109.3 kDa) was expressed in the NativeFolderTM bacterial culture medium. Bacterial pellet
was resuspended and lysed in the NativeLyserTM buffer and the cell-free extract purified on a Ni-NTA affinity
column. M: Mark12TM MW Ladder, CFE: Cell-Free Extract, FT: Flow-through W: Wash, Ei: Elution.
116.3
97.4
66.3
55.4
36.5
31.0
21.5
14.4
6.0
Protein 3
(109.3 kDa)

o  NativeFolderTM is the only bacterial culture medium on the market that
specifically promotes the expression of soluble proteins.
o  NativeFolderTM is very easy to use. User simply adds di-water to Mix A and
Mix B, sterilizes the mixture and grows the bacteria.
o  NativeFolderTM solubilizes proteins without any modification or additional
genetic engineering of the target protein (addition of tags, fusion proteins,
re-cloning, PCR, etc.).
o  NativeFolderTM efficacy in solubilizing recombinant proteins has been
proven with multiple proteins, as large as 110 kDa, including wild-type
proteins, mutants, fusion proteins, human proteins, etc.
o  NativeFolderTM formulation is the result of a 15-years R&D experience in
the solubilization of proteins in E. coli.
What Makes NativeFolderTM Unique?

o  On one hand, because of its reasonable cost, a small investment in
NativeFolderTM bears virtually no risk to both academic and industrial
researchers. It takes as little as two days to effectively test NativeFolderTM.
o  On the other hand, NativeFolderTM can be extremely rewarding because it
can save researchers weeks and months of experimenting with the:
o  Renaturation of inclusion bodies
o  Learning, setting up and optimizing the expression in news hosts
o  Scaling up
o  Controlling cost
o  Optimizing yield
o  Controlling batch variability
o  Training personnel and purchasing new equipment
Why Should You Use NativeFolderTM

Expression of Soluble Proteins with NativeFolderTM

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