bioaffinity chromatography by nuhman10


									                                           Bioaffinity chromatography

Bioaffinity chromatography (chapter 6)

-   Separation based on specific reversible interaction
    of proteins with ligands

-   Ligands are covalently attached to solid support (gel

-   Chromatography on a bioaffinity matrix retains
    proteins with interaction to the column-bound

-   Proteins bound to a bioaffinity column can be eluted
    in two ways:

    1.   Biospecific elution: inclusion of free ligand in
         elution buffer which competes with column-
         bound ligand

    2.   Aspecific elution: change in pH, salt, etc. which
         weakens interaction protein with column-bound

Because of specificity of the interaction, bioaffinity
chromatography can result in very high purification in a
single step (10 - 1000-fold !!)
                                                Bioaffinity chromatography

Protein-ligand interaction

                E       +       L           EL

When ligand is bound to solid support:

               E    +       LM             E-LM

Binding of ligand to column should not interfere to much
with interaction with protein:

              10-4 M        <       K'd <   10-10 M

Amount of enzyme retained on column (E-LM)
dependent on:
    -  K'd (interaction is to weak if K'd > 10-4 M)
    - Concentration enzyme in extract [E]
    - Degree of substitution of matrix [LM]

Biospecific elution with competitive ligand I dependent
    -    Concentration of competitive ligand [I]
    -    Interaction of competitive ligand with E-LM: Ki
                                                Bioaffinity chromatography


Small (bio)molecules : substrates, inhibitors, cofactors

Large (bio)molecules: antibodies, receptors, proteins

Ligand                    Specificity                                 .
NAD, NADP                 Dehydrogenases
5’-AMP                    NAD-dependent enzymes
2',5'-ADP                 NADP+-dependent enzymes
Glutathione               Glutathione-S-transferase (fusion proteins)
Chitin                    Chitin binding protein (fusion proteins)
Amylose                   Maltose binding protein (fusion proteins)
Blue B                    Kinases, dehydrogenases
Blue F3G-A                NAD+-dependent enzymes
Red HE-3B                 NADP+-dependent enzymes
Green A                   CoA proteins, HSA, dehydrogenases
Lysine                    rRNA, ds-DNA, plasminogen
Arginine                  Fibronectin, prothrombin
Benzamidine               Serine proteases
Calmodulin                Kinases
Gelatin                   Fibronectin
Polymyxin                 Endotoxins
Heparin                   Lipoproteins, DNA, RNA
Lectins, concanavalin A   Polysaccharides, glycosylated proteins
Antibodies (IgG)          Antigen (protein, etc)
Protein A                 Fc antibody fragments
Protein G                 Antibodies
Poly U, oligo-dT          Poly(A)+ mRNA
                                        Bioaffinity chromatography

How to make your own bioaffinity matrix.

1.   Activation of matrix.
2.   Covalent attachment of spacer (linker) group with
     reactive end group.
3.   Attachment of ligand to spacer

Activation of matrix

A common procedure for sugar-based matrices is
activation with cyanogen bromide:
                                                              Bioaffinity chromatography


                      N                         C   N     L
                      H                         O   H

  Bead                          Spacer                            Ligand


           a) Sepharose   NH (CH 2)6 NH 2

           b) Sepharose   NH (CH 2)5 COOH
           c) Sepharose NH (CH 2)5 CO O N

           d) Sepharose O CH 2 CH CH 2 O (CH 2)4 O CH 2 CH CH 2
                                 OH                       O
                                        Bioaffinity chromatography

Coupling of ligand

-   Chemistry of coupling reaction
       (e.g. coupling of –COOH to –NH2 groups by
       carbodiimide-catalyzed water extraction)

-   Residues on ligand essential for binding to protein

-   Blocking excess reactive groups
                                          Bioaffinity chromatography

Example 1: Isolation of an NAD-dependent
           dehydrogenase from a crude extract on

      1. LOADING

      Column: NAD-agarose
      Sample: crude extract with NAD-dependent
      dehydrogenases 2. WASHING

                Wash with high-salt buffer to remove all
                non-bound proteins from column.
                Proteins with strong interaction with NAD
                (Kd = 10-4 – 10-10 M) remain bound to the

                         3. ELUTION

                         Bio-specific elution of bound
                         proteins with buffer containing 1 –
                         10 mM NAD
                         Free NAD competes with column
                         bound NAD

                         (Non-specific elution by salt, pH,
                          temp. change)
                                                                                 Bioaffinity chromatography

Example 2: Isolation of an NAD-dependent
           dehydrogenase from a crude extract with
           dye-affinity chromatography

Many polycyclic textile dyes are competitive inhibitors for
NAD(P) dependent redox enzymes --> they can be used
as ligands for affinity purification.
                           O        NH 2
                                           SO3Na                      SO3Na

                           O        NH             NH           N
                                               SO3Na            O
                               Cibracon Blue F3G-A

                  NaO 3S                                                 SO3Na

                           N                                         N
               NaO 3S      N                                         N      SO3Na

                               OH                               HO

               NaO 3S      NH                                        NH      SO3Na
                                    N                       N
                           N            NH          NH               N
                                    N                       N
                           O                                         OH
                                 Procion Red HE 3B

-   Cheap
-   Much more stable than NAD or AMP agarose (dyes
    are not substrates
-   Different dyes have different specificities. Interaction
    cannot be predicted.
                                           Bioaffinity chromatography

Example 3: Fusion proteins with an affinity tag

1.   Insertion of gene for protein to be purified in a vector
     containing coding sequence for an ‘affinity tag’ (e.g.
     chitin-binding (CBP) tag, glutathione S-transferase
     (GST) binding tag). Insertion in correct reading
     frame results in fusion protein.
     Amino acid sequence recognized by rare-cutting
     endoprotease or intein sequence can be inserted.
     Strong promoter ensures high expression.

            Intein-CPB fusion-expression vector
                                         Bioaffinity chromatography

2.   Expression in E. coli results in high amounts of
     fusion protein; after chromatography on affinity
     matrix (glutathione-sepharose, chitin beads) fusion
     protein is specifically retained.
                                        Bioaffinity chromatography

3.   After on-column splicing (endonuclease, induction of
     intein-mediated splicing) mature protein can be
     eluted in almost pure form.

Purification of an NADH-reductase from a CBP-intein-NADH
            reductase triple fusion protein by affinity
        chromatography on chitin beads and on-column

4.   Regeneration of the column by removal of tag (wash
     with glutathione; guanidinium chloride.

An affinity tag is very helpful for purification of
proteins without enzymatic activity (e.g. regulatory

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