Single-column purification of free recombinant proteins - Genetik
Transcript of Single-column purification of free recombinant proteins - Genetik
Single-column purification of free
recombinant proteins using a self-
cleavable affinity tag derived from a
protein splicing elementprotein splicing element
Shaorong Chong et al.,
Gene 192 (1997), 271-281
Outline1. Introduction
2. Objectives
3. Results
3.1 Construction of MYB fusion protein and characteristics of the affinity tag
3.2 Thiol-induced cleavage of free MYB fusion protein3.2 Thiol-induced cleavage of free MYB fusion protein
3.3 Thiol-induced cleavage of immobilized MYB fusion protein
3.4 Construction of the pCYB vector
3.5 Expression and purification of HhaI methylase and other proteins
3.6 In vitro labeling
4. Summary
5. Take-Home-Lesson
Conventional affinity chromatography
target affinity
Disadvantages:
• protease may not be only linker-specific
• linker-sequence may inaccessible for the protease
• cleavage is depend on temperature and pH
• additional separation of the target protein from the
affinity tag and the proteases
target affinity
protein linker tagMelanie Gertz, BCII Seminar (2011)
Noval affinity chromatography
• Using a self-cleaving linker � intein
• Modification of the intein:
Substitution Asn454 to AlaSubstitution Asn454 to Ala
� unable to splice
� only N-terminal cleavage
• Initialize the cleavege mechanism
with reactive thiols (β-ME, DTT, cysteine)
Wolfgang Schumann,
Gentechnologie (2011)
Objectives
• Influence of pH, temperature, salt concentration and
detergents on cleavage of MYB fusion protein
• Construction of an expression vector and expression and
purification of various target proteins
3.1 Construction and of MYB fusion protein and characteristics of the affinity tag
pMYB129:
MBP: maltose-binding protein from E. coli
modified intein: Sce VMA with Asn454Ala substitution modified intein: Sce VMA with Asn454Ala substitution
CBD: chitin-binding domain from C-terminal region of chitinase A1 from Bacillus circulans
Caracteristics of the CBD affinity tag:
MYB fusion protein binds on chitin beads
• no effect on the binding with 1 M NaCl, 0,1% Triton X-100, Tween 20, 1 mM EDTA
• MYB released from beads with 6 M guanidinium or >0,5% SDS
3.2 Thiol-induced cleavage of free MYB fusion protein
Cleavage under several conditions:
Nucleophiles, incubation time, pH, NaCl concentrations,
presence of Triton X-100, Tween 20
(30 mM DTT)
3.2 Thiol-induced cleavage of free MYB fusion protein
Conclusion:
• MYB fusion protein stable after incubation at 25 - 50°C and at
pH 5,5 – 9,0 (4°C)
• Cleavage with DTT, β-ME and cystein was efficient even in
presence of 2 M NaCl, 0,1% Triton X-100 or Tween 20
• Reaction most efficient with DTT at high pH
• Cleavage with hydroxylamine was less efficient
3.3 Thiol-induced cleavage of immoblized MYB fusion protein
• Only MBP was eluated � cleavage has no effect on the binding
of CBD
• After washing with 2% SDS, only YB was eluated
• 90% yield of MBP after incubated with 30 mM DTT for 16 h at 4°C• 90% yield of MBP after incubated with 30 mM DTT for 16 h at 4°C
3.5 Expression and purification of HhaI methylase and other proteins
• Efficient purification of prokaryotic proteins
• Less efficient purification of eukaryotic proteins
3.6 In vitro labeling
1. Expression of MBP and BamHI
by the pCYB vector
2. Purification with chitin-column;
cleavage is DTT-inducedcleavage is DTT-induced
3. Incubation with L-[S35]cysteine
after eluation � labeled
4. Negative control: Purification of free MBP on amylose resin
and incubation with L-[S35]cysteine � not labeled
4 & 5: visualized by autoradiography
Summary• Cleavage of a modified intein with a reactive nucleophile (thiol)
• Cleavage has no influence on the binding of CBD affinity tag
• Purification is efficient over a wide range of pH and temperature
and in presence of high salt concentrations and severel
detergents (Triton X-100, Tween 20)
• Purification most efficient with DTT at basic pH
• Expression and purification of various recombinant proteins
possible (eukaryotic proteins less efficient) by using the pCYB
vector
• Possibility of in vitro labeling
Take-Home-Lesson
Fusion proteins including a modified intein allow
purification with a single column step and under
various conditions induced by only cheap chemicals.