Single-column purification of free recombinant proteins - Genetik

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Single-column purification of free recombinant proteins using a self- cleavable affinity tag derived from a protein splicing element protein splicing element Shaorong Chong et al., Gene 192 (1997), 271-281

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

Recombinant protein expression

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)

Spicing of the Sce VMA intein

Chong et al., Biolog. Chem. 271 (1996), 271-281

Cleavage with the modified intein

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

3.4 Construction of the pCYB vector

3.5 Expression and purification of HhaI methylase and other proteins

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

3.6 In vitro labeling

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.

Diskussion

• Was versteht man unter Protein-Splicing?

• Wie kann man Protein-Splicing bei der • Wie kann man Protein-Splicing bei der

Produktion von rekombinanten Proteinen

einsetzen?