Transcript Expression Validation

Protein Expression in E. coli using
pEX Expression Vectors
• Validation Data
5 New E. coli Expression Vectors
Please see vector maps at http://www.origene.com/cdna/TrueORF/DestinationVector.aspx
then click on “Bacterial expression” tab
Protein Expression in E. coli N-tagged vector
4 different mammalian ORFs
was cloned into the pEX-N-HisGST vector.
GST-fused proteins were
expressed at high levels with
all constructs.
Protein
Expression
in pEX-C-His
Vector Vector
Protein
Expression
in pEX-C-His
MW (kDa)
1
2
191
97
64
51
BIN1
39
28
1. pEX-C-His-Bin1 (NM_139351) no IPTG
2. pEX-C-His-Bin1 (NM_139351) with IPTG
The ORF of Bin1 was cloned into
pEX-C-His vector and expressed in
BL21(DE3) cells.
Total protein lysates were loaded on
the SDS-PAGE gel.
Highly Efficient Protein Expression
pEX-N-His-GST vector shows
comparabe high expression level of
GST with pGEX-2T vector.
The Fusion Tag Can Be Cleaved
A purified GST fusion protein was
incubated with TEV protease at
30oC for the indicated period of
time. The recombinant protein and
GST tag were separated by TEV
cleavage (C).
pEX-1 for Untagged Expression
When no-tag is desired for the
recombinant protein, pEX-1 is the
vector of choice, appending only
two amino acid (T and R) to the
C-terminus of the protein.
Easy TrueORF Shuttling into pEX
37,000 TrueORF clones can be
easily shuttled from entry vector into
E. coli pEX vectors.
*
The entry and destination vectors
are digested with two rare cutters:
Sgf I and Mlu I or other specified
enzymes.
For pEX-1 and pEX-C-His vectors
use BseR I and MluI for subcloning.
Order TrueORF in E. coli Vectors
For all destination vectors, there are two options:
1. Purchase both the TrueORF in pCMV6-Entry vector
and the desired destination vector and follow the
simple shuttling protocol.
2. Ask OriGene experts to perform the shuttling.
OriGene will provide highly purified ready-to-use
plasmids.