Sucrose → Imidazole-4-acetic acid

HRID 1255389Notes

Reaction equation

HRID 1255389 reaction equation
CC(S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O)C.C(O)[C@H]1[O:22][C@H:21]([O:23][C@]2(CO)O[C@H](CO)[C@@H](O)[C@@H]2O)[C@H:20](O)[C@@H](O)[C@@H]1O.[CH2:39]([N:50]([CH2:55]C(O)=O)CC(O)=O)[CH2:40][N:41](CC(O)=O)CC(O)=O>P([O-])([O-])([O-])=O.[Na+].[Na+].[Na+]>[CH:40]1[N:41]=[CH:55][NH:50][C:39]=1[CH2:20][C:21]([OH:23])=[O:22]

Participants

5

Reactant 3

HCID 5988SucroseHCID 6049Ethylenediaminetetraacetic AcidHCID 656894isopropyl beta-D-thiogalactopyranoside

Product 1

HCID 96215Imidazole-4-acetic acid

Reagents, catalysts & solvents 1

HCID 24243Trisodium PhosphateSolvent

Procedure

The coding sequence of the VHH was subcloned in vector pET 22 using the NcoI and NotI restriction sites according to the manufacturer's instructions (Novagen, Darmstadt, Germany). Transformed E. coli BL 21 (DE3) cells expressed VHHs in the periplasm after induction by IPTG 1 mM for 18 hours at 15° C. Periplasmic extracts were obtained by spheroplasting cells, suspended in 50 mM sodium phosphate buffer pH 8 containing 20% sucrose and 1 mM EDTA, and hydrolysing the peptidoglycan with 5 mg/ml lysozyme for 20 min at 4° C., in the presence of protease inhibitors (Complete™, Boehringer Mannheim, Germany). The suspension was then centrifuged 2 min at 10,000 rpm. The supernatant corresponding to the periplasmic extract was kept at 4° C. Purified VHHs were obtained by IMAC using a chelating agarose column charged with Ni2+ (Superflow Ni-NTA, Qiagen Ltd, UK) according to manufacturer's instructions. Purified VHH were dialysed against PBS and the protein content was measured using the Bradford reagent. The purity of the final preparation was evaluated by SDS-PAGE with Coomassie staining and by Western blot.

Workup

  1. customfor 18 hours
  2. customat 15° C
  3. customPeriplasmic extracts were obtained
  4. waitThe suspension was then centrifuged 2 min at 10,000 rpm
  5. extractionThe supernatant corresponding to the periplasmic extract
  6. customwas kept at 4° C
  7. customPurified VHHs

Sources & evidence

ORD record
ord-a0cdd2011d9e4419aab9bb6f3851f95b
Source dataset
uspto-grants-2013_06
Patent number
US08460888B2

Reaction notes

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