Thiamine → L-Hydroxyproline

HRID 399376

Reaction equation

HRID 399376 reaction equation
CC(S[C@@H]1[O:10][C@H](CO)[C@H](O)[C@H](O)[C@H]1O)C.[NH:16]1[CH2:23][CH2:22][CH2:21][C@H:17]1[C:18]([OH:20])=[O:19].CC1(C)S[C@@H]2[C@H](NC([C@H](N)C3C=CC=CC=3)=O)C(=O)N2[C@H]1C(O)=O.O=C[C@@H]([C@H]([C@@H]([C@@H](CO)O)O)O)O.CC1[N+](CC2C=NC(C)=NC=2N)=CSC=1CCO>>[NH:16]1[CH2:23][C@H:22]([OH:10])[CH2:21][C@H:17]1[C:18]([OH:20])=[O:19]

Participants

6

Reactant 5

HCID 1130ThiamineHCID 6249Ampicillin4×HCID 107526(2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanalHCID 145742L-Proline2×HCID 656894isopropyl beta-D-thiogalactopyranoside2×

Product 1

HCID 5810L-Hydroxyproline

Conditions

Temperature
37 °C

Procedure

A plasmid (pHuCol, FIG. 2) encoding the gene sequence of human Type I (α1) collagen (FIGS. 3A and 3B (Seq. ID No. 1placed behind the isopropyl-β-D-thiogalactopyranoside (IPTG)-inducible tac promotor and also encoding β-lactamase is transformed into Escherichia coli proline auxotrophic strain NM519 (pro-) by standard heat shock transformation. Transformation cultures are plated on Luria Broth (LB) containing 100 μg/ml ampicillin and after overnight growth a single ampicillin-resistant colony is used to inoculate 5 ml of LB containing 100 μg/ml ampicillin. After growth for 10-16 hours with shaking (225 rpm) at 37° C., this culture is used to inoculate 1 L of M9 minimal medium (M9 salts, 2% glucose, 0.01 mg/mL thiamine, 100 μg/ml ampicillin, supplemented with all amino acids at 20 μg/mL except proline which is supplemented at 12.5 mg/L) in a 1.5 L shaker flask. After growth at 37° C., 225 rpm, for 15-20 hours post-inoculation, the optical density at 600 nm is constant at approximately 0.5 OD/ml. The cells are harvested by centrifugation (5000 rpm, 5 minutes), the media decanted, and the cells resuspended in 1 L of M9 minimal media containing 100 μg/ml ampicillin, 0.08M L-hydroxyproline, and 0.5M NaCI. Following growth for 1 hour at 37° C., 225 rpm, IPTG is added to 1 mM and the cultures allowed to grow for an additional 5-15 hours. Cells are harvested by centrifugation (5000 rpm, 10 minutes) and lysed by mechanical disruption. Hydroxyproline-containing collagen is purified by ammonium sulfate fractionation and column chromatography.

Workup

  1. waitAfter growth at 37° C., 225 rpm, for 15-20 hours post-inoculation, the optical density at 600 nm is constant at approximately 0.5 OD/ml
  2. customare harvested by centrifugation (5000 rpm, 5 minutes)
  3. customthe media decanted
  4. additioncontaining 100 μg/ml ampicillin, 0.08M L-hydroxyproline, and 0.5M NaCI
  5. customfor 1 hour
  6. customat 37° C.
  7. addition225 rpm, IPTG is added to 1 mM
  8. waitto grow for an additional 5-15 hours
  9. customare harvested by centrifugation (5000 rpm, 10 minutes)
  10. additionHydroxyproline-containing collagen
  11. customis purified by ammonium sulfate fractionation and column chromatography

Sources & evidence

ORD record
ord-68ce6754c7ad4dd2978c5a8eea38de83
Source dataset
uspto-grants-1998_10
Patent number
US05821089

Linked notes

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