Preparation of Isotopically Labeled Recombinant Fragments of Fibronectin for Functional and Structural Study by Heteronuclear Nuclear Magnetic Resonance Spectroscopy - Methods in Molecular Biology: Extracellular Matrix Protocols - page 59

2. Materials
All unlabeled reagents were standard laboratory reagent grade; all solutions
were prepared using milli-Q water. 15 N-ammonium sulphate (99 At%), U-[ 13 C]-
glucose (99 At%) and 13 C-methanol (99 At%) were obtained from CK Gas
Products, (Finchampstead, UK). The zeroing gel was obtained from Mettler-
Toledo (Leicester, UK). Polypropylene glycol 1025 was obtained from Merck
(Poole, UK). A 2-L glass fermentation vessel was obtained from Electrolab
Ltd. (Tewkesbury, UK) with fittings for variable-speed impeller, temperature
control, adjustable sterile air supply, air outlet fitted with a condenser, and with
ports for pH, dissolved-oxygen (dO 2 ), and temperature probes, acid-, base-, and
nutrient-feed lines, and for inoculation and sampling. The fermenter was oper-
ated using a controller (Electrolab Ltd.) with acid and base pumps for feedback
pH control, feedback temperature control, impeller-speed control, and vari-
able-speed nutrient-feed pump ( Fig. 2 ). The chart recorder was obtained from
Kipp & Zonen (St. Albans, UK).

2.1. Seed Culture
1. Biotin solution (per 100 mL): Dissolve 0.2 g D-biotin in 10 m M sodium hydrox-

ide. Filter sterilize and store at 4 ° C for up to 3 mo.

2. Buffered Minimal Glucose (BMGc) pH 6.0 (per 100 mL): Dissolve 0.34 g yeast

nitrogen base without amino acids or ammonium sulphate (Difco, UK), 1 g
ammonium sulphate, 1 g D-glucose, 1.19 g potassium dihydrogen phosphate,
0.21 g di-potassium hydrogen phosphate, and 0.2 mL biotin solution in water.
Filter sterilize and store at 4 C for up to 1 month. Use 15 N-ammonium sulphate

and/or U-[ 13 C]-glucose instead as appropriate ( see Note 1 ).

°

2.2. Preparation of the Vessel
1.

Basal Salts Medium (BSM) pH 6.0 (per liter): Dissolve 0.93 g calcium sulphate,
14.9 g magnesium sulphate heptahydrate, 25.0 g potassium hydroxide (corro-
sive), and 26.7 mL orthophosphoric acid (85 wt % - corrosive) in water. Auto-
clave and adjust pH in the fermentation vessel ( see Note 2 ).

2. BSM pH 3.0 (per liter): Dissolve 0.93 g calcium sulphate, 14.9 g magnesium

sulphate heptahydrate, 18.2 g potassium sulphate, and 26.7 mL orthophosphoric
acid (85 wt % - corrosive) in water. Autoclave and adjust pH in the fermentation
vessel ( see Note 2 ).

3. Pichia Trace Metals (PTM 1 ) (per liter): Dissolve 6 g copper (II) sulphate, 0.08 g

sodium iodide, 3 g manganese sulphate monohydrate, 0.2 g sodium molybdate
dihydrate, 0.02 g boric acid, 0.5 g cobalt chloride, 20.0 g zinc chloride, 65.0 g
iron (II) sulphate, 5 mL sulfuric acid (98% - corrosive) in water. Store at room
temperature for up to 1 yr; filter sterilize before use.

4. Ammonium sulphate solution: Prepare a 20 % (w/v) solution in water. Filter ster-

ilize and store at room temperature for up to 1 yr. Use 15 N-ammonium sulphate as
necessary ( see Note 3 ).