Biomedical Engineering Reference
In-Depth Information
It is possible to engineer proteins such that they
are transported through the outer membrane and
are secreted into the growth medium. This is achieved
by making use of the type I, Sec-independent secre-
tion system. The prototype type I system is the
haemolysin transport system, which requires a short
carboxy-terminal secretion signal, two translocators
(HlyB and D), and the outer-membrane protein
TolC. If the protein of interest is fused to the carboxy-
terminal secretion signal of a type I-secreted protein,
it will be secreted into the medium provided HlyB,
HlyD and TolC are expressed as well (Spreng
et al.
2000). An alternative presentation of recombinant
proteins is to express them on the surface of the
bacterial cell using any one of a number of carrier
proteins (for review, see Cornelis 2000).
Cla
I
3018
tac
promoter
Eco
R
I
3277
Pst
I
3316
Sal
I
52
Bst
X
I
264
Sac
I
377
T7 promoter
Hpa
I
2777
purification
tag sequence
1-386
Nru
I
Hin
d
III
Pvu
II
Bam
H
I
Acc
65
I
Kpn
I
Eco
R V
Bgl
II
Sma
I
Not
I
389
392
400
404
410
414
418
422
430
435
452
ori
Factor
Xa site
379-390
PinPoint
TM
Xa-1
Vector
(3351bp)
Nde
I
532
SP6
Amp
r
Fsp
I
1483
Xmn
I
1106
Pvu
I
1337
Sca
I
1225
Fig. 5.17
Structural features of a PinPoint
TM
vector.
(Figure reproduced courtesy of Promega Corporation.)
Putting it all together:
vectors with combinations of features
Many of the vectors in current use, particularly
those that are commercially available, have com-
binations of the features described in previous sec-
tions. Two examples are described here to show the
connection between the different features. The first
example is the LITMUS vectors that were described
earlier (p. 73) and which are used for the generation
of RNA probes. They exhibit the following features:
• The polylinkers are located in the
lac
Z
• The vectors are small (
<
3 kb) and with a pUC
ori
have a high copy number.
The second example is the PinPoint series of
expression vectors (Fig. 5.17). These vectors have
the following features:
• Expression is under the control of either the T7
or the
tac
promoter, allowing the user great flexibil-
ity of control over the synthesis of the cloned gene
product.
• Some of them carry a DNA sequence specifying
synthesis of a signal peptide.
• Presence of an MCS adjacent to a factor-Xa cleav-
age site.
• Synthesis of an N-terminal biotinylated sequence
to facilitate purification.
• Three different vectors of each type permitting
translation of the cloned gene insert in each of the
three reading frames.
• Presence of a phage SP6 promoter distal to the
MCS to permit the synthesis of RNA probes com-
plementary to the cloned gene. Note that the orienta-
tion of the cloned gene is known and so the RNA
probe need only be synthesized from one strand.
What is absent from these vectors is an M13 origin of
replication to facilitate synthesis of single strands for
DNA sequencing.
α
gene and
inserts in the polylinker prevent
-complementation.
Thus blue/white screening (see Box 3.2 on p. 35)
can be used to distinguish clones with inserts from
those containing vector only.
• The LITMUS polylinkers contain a total of 32
unique restriction sites. Twenty-nine of these enzymes
leave four-base overhangs and three leave blunt ends.
The three blunt cutting enzymes have been placed at
either end of the polylinker and in the middle of it.
• The vectors carry both the pUC and the M13
ori
regions. Under normal conditions the vector replic-
ates as a double-stranded plasmid but, on infection
with helper phage (M13KO7), single-stranded mole-
cules are produced and packaged in phage protein.
• The single-stranded molecules produced on helper-
phage addition have all the features necessary for
DNA sequencing (see p. 123).
α
