Biomedical Engineering Reference
In-Depth Information
EK
Site
P
BAD
term
ATG 6 x His Epitope
MCS
pBAD/His
A,B,C
4.1 kb
*Frame - dependent variations
COMMON SEQUENCE
VARIABLE SEQUENCE
GAC GAT
GAC GAT AAG
GAT
CCG AGC TCG
AGA TCT
GCA
GCT
Ala
Asp
Asp
Asp
Asp
Lys
Asp
Pro
Ser
Ser
Ars
Ser
Ala
GAC GAT
GAC GAT AAG
GAT
CGA TGG GGA TCC GAG CTC
Arg
GAG
Glu
ATC
Ile
TGC
Cys
Asp
Asp
Asp
Asp
Lys
Asp
Trp
Gly
Ser
Glu
Leu
GAC GAT
GAC GAT AAG
GAT
CGA TGG ATC CGA
CCT CGA
GAT
Asp
CTG
Leu
CAG
Gln
Asp
Asp
Asp
Asp
Lys
Asp
Arg
Trp
Ile
Arg
Pro
Arg
Enterokinase
cleavage
Fig. 5.13
Structure of a vector (pBAD/His, In Vitrogen Corporation) designed for the expression of a cloned gene as a fusion
protein containing a polyhistidine sequence. Three different variants (A, B, C) allow the insert to be placed in each of the three
translational reading frames. The sequence shaded pink shows the base sequence which is altered in each of the three vectors.
The lightly-shaded box (AGATCT) is the Bgl II site of the polylinker. Note that the initial A residue of the restriction site is at a
different point in the triplet codon in each of the three sequences.
For the cloned gene to be expressed correctly, it
has to be in the correct translational reading frame.
This is achieved by having three different vectors,
each with a polylinker in a different reading frame
(see Fig. 5.13). Enterokinase recognizes the sequence
(Asp)
4
Lys and cleaves immediately after the lysine
residue. Therefore, after enterokinase cleavage, the
cloned gene protein will have a few extra amino
acids at the N terminus. If desired, the cleavage
site and polyhistidines can be synthesized at the C
terminus. If the cloned gene product itself contains
an enterokinase cleavage site, then an alternative
protease, such as thrombin or factor Xa, with a
different cleavage site can be used.
To facilitate assay of the fusion proteins, short
antibody recognition sequences can be incorporated
into the tag between the affinity label and the pro-
tease cleavage site. Some examples of recognizable
