Biomedical Engineering Reference
In-Depth Information
Cleave
Target Gene
CBD
Strip with
SDS
T7 promot
er
Load
F.T.
Wash
+
DTT
Elute
MCS
I
ntein
Cloning and
expression
-IP
TG
+
I
PT
G
CBD
4
°
C
o.n.
Target Protein
I
ntein
Intein
Load and
wash
Chitin
kDa
212
158
116
97.2
66.4
55.6
42.7
36.5
Inducible cleavage
+DTT at 4
°
C
Precursor
Intein - CBD
MBP
Elute
Target Protein
26.6
20.0
12345678910
Lane 1: Protein Marker.
Lane 2: Crude extract from uninduced cells.
Lane 3: Crude extract from cells, induced at 15
°
C for 16 hours.
Lane 4: Clarified crude extract from induced cells.
Lane 5: Chitin column flow through (F.T.).
Lane 6: Chitin column wash.
Lane 7: Quick DTT wash to distribute DTT evenly throughout the chitin column.
Lanes 8-9: Fraction of eluted MBP after stopping column flow and inducing a self-cleavage reaction at 4
°
C overnight.
Lane 10: SDS stripping of remaining proteins bound to chitin column (mostly the cleaved intein-CBD fusion).
Fig. 5.15
Purification of a cloned gene product synthesized as a fusion with an intein protein. (Figure reproduced courtesy of New
England Biolabs.)
systematically mutated a cluster of surface amino
acid residues. Residues 30 and 62 were converted
to histidine and the modified ('histidine patch')
thioredoxin could now be purified by affinity chro-
matography on immobilized divalent nickel. An
alternative purification method was developed by
Smith
et al.
(1998). They synthesized a gene in
which a short biotinylation peptide is fused to the N
terminus of the thioredoxin gene to generate a new
protein called BIOTRX. They constructed a vector
carrying the BIOTRX gene, with an MCS at the C ter-
minus, and the
bir
A gene. After cloning a gene in
the MCS, a fused protein is produced which can be
purified by affinity chromatography on streptavidin
columns.
An alternative way of keeping recombinant pro-
teins soluble is to export them to the periplasmic
space (see next section). However, even here they
may still be insoluble. Barth
et al.
(2000) solved this
problem by growing the producing bacteria under
osmotic stress (4% NaCl plus 0.5 mol/l sorbitol) in
the presence of compatible solutes. Compatible
solutes are low-molecular-weight osmolytes, such
as glycine betaine, that occur naturally in halophilic
bacteria and are known to protect proteins at high
salt concentrations. Adding glycine betaine for the
cultivation of
E. coli
under osmotic stress not only
allowed the bacteria to grow under these otherwise
inhibitory conditions but also produced a periplas-
mic environment for the generation of correctly
folded recombinant proteins.
Vectors to promote protein export
Gram-negative bacteria such as
E. coli
have a com-
plex wall-membrane structure comprising an inner,
cytoplasmic membrane separated from an outer
membrane by a cell wall and periplasmic space.
