Intein Mediated Purification

Intein Mediated Purification

Download Free online IMPACT ™ -CN Protein Purification System Now Featuring Fusion to C- or N- Terminus of the Target Protein Manual PDF

Download Free Online NEB Impact TWIN Manual

MPACT (
Intein
Mediated
Purification with an
Affinity
Chitin-binding
Tag) is a novel
protein purification system which utilizes the inducible self-cleavage activity of a
protein splicing element (termed intein) to separate the target protein from the affinity
tag. It distinguishes itself from all other purification systems by its ability to purify, in
a single chromagraphic step, a native recombinant protein without the use of a
protease. The concept of IMPACT purification has evolved from our studies of the
protein splicing mechanism [Appendix II &
IV (1
6)]. The IMPACT-CN System utilizes
an intein (454 amino acid residues) from the
Saccharomyces cerevisiae
VMA1 gene. A
target protein is fused to a self-cleavable
intein tag in which a chitin binding domain
allows affinity purification of the fusion
precursor on a chitin column. In the
presence of thiols such as DTT,
β
mercaptoethanol or cysteine, the intein
undergoes specific self-cleavage which
releases the target protein from the chitin-
bound intein tag resulting in a single-
column purification of the target protein
(Figure 1, 2).
Figure 1:
A schematic illustration of the
IMPACT-CN System.
Intein
Tag
Target
Protein
Intein
Tag
Target
Protein
Target
Protein
Target
Protein
Target Gene
MCS
N-terminal
C-terminal
Target Gene
MCS
Intein Tag
N-terminal
C-terminal
Intein Tag
Load &
Wash
Inducible Cleavage
+DTT at 4
°
C
C-terminal Fusion
N-terminal Fusion
T7 Promoter
T7 Promoter
Chitin
Chitin
Elute &
Dialyze
Elute
The IMPACT-CN System contains expression vectors (pTYB vectors) which
allow the fusion of the cleavable intein tag to either the C-terminus (pTYB1 and pTYB2,
C-terminal fusion) or N-terminus (pTYB11and pTYB12, N-terminal fusion) of a target 3 protein. This flexibility in fusion protein construction maximizes the probability of successful expression and purification of a target protein. To allow the cloning of the same amplified target gene in either fusion construction, the same or compatible restriction sites are designed in the multiple cloning region of pTYB2 and pTYB12 vectors. pTYB1 and pTYB11 vectors, on the other hand, allow the cloning of a target gene immediately adjacent to the intein cleavage site. This results in the  purification of a native target protein without any vector-derived extra residues after the cleavage. A
detailed comparison between the N-terminal and C-terminal fusion with the intein tag is described in Appendix I. The pTYB vectors use a T7 promoter-driven system (11) to achieve high levels of expression and tight transcriptional control in E. coli
.
The IMPACT-CN System provides 4 major advances over other expression systems:
(i) rapid and simple purification of a native protein without extra vector-derived
residues; (ii) high affinity of the chitin binding domain to allow more stringent
washing conditions and reduce non-specific binding; (iii) easy separation of the
target protein from the affinity tag without the use of a separate, expensive
protease; and (iv) the only protein expression and purification system to allow specific
C-terminal labeling of a target protein and the Intein-mediated Protein Ligation (IPL)
(see Appendix IV

Spread the love

Leave a Reply

Your email address will not be published. Required fields are marked *