Protein Protocols Protein Protocols

366 Haugland and Bhalgat
sidered, instead. A protocol for radioiodination of streptavidin using IODO-BEADS ®
(6) is described in Subheading 3.2. Some other methods that have been developed
include the iodogen method (7,8), see also Chapter 115, the Bolton–Hunter reagent
method (9), and a few that do not involve direct iodination of tyrosine residues (10–13).
Streptavidin–drug conjugates are also candidates for therapeutic agents. Synthesis of a
streptavidin-drug conjugate involves making a chemically reactive form of the drug
followed by its conjugation to streptavidin. The synthetic methodology thus depends
on the structure of the specific drug to be conjugated (14–16).
The avidin-biotin interaction can also be exploited for affinity chromatography; however,
there are limitations to this application, because a biotinylated protein captured
on an avidin affinity matrix would likely be denatured by the severe conditions required
to separate the high-affinity avidin–biotin complex. On the other hand, an avidin affinity
matrix may find utility in the removal of undesired biotinylated moieties from a
mixture or for the purification of compounds derivatized with 2-iminobiotin. The biotin
derivative 2-iminobiotin has reduced affinity for avidin, and its moderate binding to
avidin at pH 9.0 is greatly diminished at pH 4.5 (17). Another approach to reducing
the affinity of the interaction is to denature avidin to its monomeric subunits. The
monomeric subunits have greatly reduced affinity for biotin (18). We describe here a
protocol for preparing native (19) and monomeric avidin matrices (20). Modified
streptavidins, hybrids of native and engineered subunits with lower binding constants,
have been prepared that may also be suitable for affinity matrices (21). A new form of
monovalent avidin, nitrated at the tyrosine located at three of the four biotin binding
sites, has recently been available from Molecular Probes. The binding affinity of this
modified avidin (22), called CaptAvidin biotin-binding protein, is lower than for the
native protein. At pH 4 CaptAvidin biotin-binding protein associates with biotin with a
K a of ~10 9 M –1 ; if needed, the complex can be completely dissociated at pH 10.0. This
property makes CaptAvidin biotin-binding protein the ideal ligand for affinity matrices
suitable for isolation and purification of biotinylated compounds. A nitration protocol
is described in Chapter 64.
2. Materials
2.1. Conjugation to Antibodies, Enzymes or Oligonucleotides
1. Avidin (mol wt = 66,000).
2. Antibody, enzyme, peptide, protein, or thiolated oligonucleotide to be conjugated to avidin.
3. Succinimidyl 3-(2-pyridyldithio)propionate (SPDP; mol wt = 312.36) (see Note 3).
4. Succinimidyl trans-4-(N-maleimidylmethyl)cyclohexane-1-carboxylate (SMCC; mol wt
= 334.33).
5. Dithiothreitol (DTT; mol wt = 154.24).
6. Tris-(2-carboxyethyl) phosphine (TCEP; mol wt = 286.7).
7. N-ethylmaleimide (NEM; mol wt = 125.13)
8. Anhydrous dimethyl sulfoxide (DMSO) or anhydrous dimethylformamide (DMF).
9. 0.1 M Phosphate buffer: Contains 0.1 M sodium phosphate, 0.1 M NaCl at pH 7.5. Dissolve
92 g of Na2HPO4, 21 g of NaH2PO4·H2O, and 46.7 g of NaCl in approx 3.5 L of
distilled water and adjust the pH to 7.5 with 5 M NaOH. Dilute to 8 L. Store refrigerated.
10. 1 M Sodium bicarbonate (see Note 4). Dissolve 8.4 g in 90 mL of distilled water and
adjust the volume to 100 mL. A freshly prepared solution has a pH of 8.3–8.5.