Protein Protocols Protein Protocols

1042 Kipriyanov
9. For analytical size-exclusion chromatography, apply 50 µL of the concentrated preparation
of SCA to a Superdex HR10/30 column. Perform gel filtration at 4°C, monitor the
UV-absorption of effluent at 280 nm, and, if necessary, collect 0.5-mL fractions.
10. For long-term storage, stabilize purified antibody fragments by adding Human serum
albumin (HSA) to a final concentration of 10 mg/mL. Store the sample at –80°C (see Note 21).
3.5. Analysis of Binding Properties of SCAs in ELISA
1. Coat 96-well ELISA microplates with 100 µL/well of a 10 µg/mL solution of antigen in
50 mM sodium carbonate–bicarbonate buffer, pH 9.6, overnight at 4°C.
2. Wash the wells 5× with PBS–Tween 20 and incubate with 200 µL of milk–PBS at room
temperature for 2 h.
3. Load 100 µL of purified SCA at various dilutions in milk–PBS into the wells and incubate
for another 2 h at room temperature.
4. Wash the wells 10× with PBS–Tween 20, add 100 µL of a 14000 dilution of mAb 9E10
in milk–PBS, and incubate the plates for 1 h at room temperature (see Note 22).
5. Wash the wells 10× with PBS–Tween 20, add 100 µL of 14000 dilution of goat antimouse
IgG-peroxidase conjugate in milk–PBS, and incubate the plates for 1 h.
6. Wash the wells 10× with PBS–Tween 20, add 100 µL of a TMB peroxidase substrate, and
incubate at room temperature for 20–40 min. Read the absorbance of the colored product
at 655 nm on a Microplate Reader.
4. Notes
1. Both XL1-Blue and RV308 are suitable hosts for expression of antibody fragments in
shake-flask bacterial cultures. XL1-Blue has the following advantages: electrocompetent
bacterial cells are commercially available (Stratagene) and standard DNA isolation protocols
yield in pure DNA preparations for restriction digests and sequencing. However,
RV308 is more robust fast growing strain suitable for high-cell density fermentation (36).
Moreover, unlike for XL1-Blue, no leakage of antibody fragments into the culture
medium was observed for RV308.
2. LB (Luria Bertani) broth can also be used. However, we observed that the simple substitution
of LB for somewhat richer 2YT medium gave an essential increase in the yield of
soluble antibody molecules.
3. 2YTSA medium is prepared directly before use by dissolving 137 g of sucrose powder in
1 L of sterile 2YT medium containing 0.1 g/L of ampicillin.
4. The protocols were established for vectors pHOG21 (25) and pSKK (9) designed for
periplasmic expression of single recombinant product.
5. All DNA manipulations and transformation experiments are performed according to standard
cloning protocols (34).
6. Direct induction without medium change is recommended for production of scFv and
other SCA in predominantly monomeric form. However, the change of medium and
induction of antibody synthesis in bacteria under osmotic stress significantly increases the
yield of recombinant product, although these conditions promote the formation of
domain-swapped dimers (9).
7. This concentration of IPTG was found to be optimal for vectors containing the SCA gene
under the control of wt lac promoter/operator such as pHOG21 (25) or pSKK (9). Nevertheless,
performing small-scale experiments to optimize the induction conditions is recommended
for each vector.
8. For XL1-Blue either due to the leakiness of the outer membrane or due to the partial cell
lysis, a significant fraction of antibody fragments is found in the culture medium. There-