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Affinity Processing of Cell-Containing Feeds 249
of microbial (9) and mammalian (7,8) cells, inclusion bodies (10) and
mitochondria (11). The use of monolithic cryogel columns will be illustrated
using the example of direct capture of extracellularly expressed histidine-tagged
protein from the fermentation broth (see Subheading 3.3.) and separation of
two different cell types, namely T and B lymphocytes (see Subheading 3.4.).
2. Materials
2.1. Coupling IMAC Ligand, Iminodiacetic Acid
1. Na 2 CO 3 , 0.5 M.
2. Na 2 CO 3 ,1M.
3. Iminodiacetic acid (IDA) solution, 0.5 M, in 1MNa 2 CO 3 , pH 10, 0.5 M CuSO 4 .
4. Ethylenediaminetetraacetic acid (EDTA), 0.1 M, pH 7.6.
2.2. Coupling Affinity Ligand, Protein A
1. Na 2 CO 3 , 0.2 M.
2. Ethylenediamine solution, 0.5 M, in 0.2 M Na 2 CO 3 .
3. Sodium phosphate buffer, 0.1 M, pH 7.2.
4. Glutaraldehyde solution, 5% v/v, in 0.1 M sodium phosphate buffer, pH 7.2.
5. Protein A solution, 1.6 mg/ml, in 0.1 M sodium phosphate buffer, pH 7.2.
6. NaBH 4 solution, 0.1 M, in sodium carbonate buffer, pH 9.2.
7. Bicinchoninic acid (BCA) solution for protein assay (Sigma).
2.3. Direct Capture of (His) 6 -Tagged Single-Chain Fv Antibody
Fragments (See Note 1)
1. Running buffer: 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
(HEPES), 200 mM NaCl, 2 mM imidazole, pH 7.
2. Elution buffer: 0.2 M imidazole in 20 mM HEPES, 200 mM NaCl, pH 7.
3. Regeneration buffer: 20 mM EDTA in 20 mM HEPES, 200 mM NaCl, pH 7.
4. Charge solution: 0.25 M CuSO 4 in distilled water.
5. Feed: The starter culture of the recombinant strain of E. coli producing extracellular
(His) 6 -tagged single-chain Fv antibody fragments was grown in Luria-Bertani (LB)
medium (tryptone 10 g; yeast extract 5 g and sodium chloride 5gin1ldistilled
water, pH 7.2) containing 0.1 mg/ml ampicillin. Expression of the target protein
was carried out in Terrific Broth (TB) medium (pancreatic digest of casein 12 g;
yeast extract 24 g; dipotassium phosphate 9.4 g and monopotassium phosphate
2.2 g in 1 l distilled water, pH 7.2) supplemented with glycerol 4 ml/l and
ampicillin 0.1 mg/ml and induced by 0.1 mM isopropyl -D-thiogalactopyranoside
at OD 600 nm = 0.5. The batch was cultivated at 37°C for 24 h with shaking at 175
rpm. The obtained fermentation broth (turbidity 18–23 units OD 450 nm ; protein =
8–10 mg/ml) was used directly with no pretreatment.
6. Stationary support: 5 ml monolithic pre-activated cryogel column produced from
polyDMAAm (Protista Biotechnology AB).