反应 #671949

ord-7428d873a35b4dd79c3f73dea37035fb

反应方程式

CC1(C)S[C@@H]2[C@H](NC(=O)[C@H](N)c3ccccc3)C(=O)N2[C@H]1C(=O)O
ampicillin
Cc1ncc(C[n+]2csc(CCO)c2C)c(N)n1
thiamine
CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O
isopropyl-β-D-thiogalactopyranoside
CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O
IPTG
CC1(C)S[C@@H]2[C@H](NC(=O)[C@H](N)c3ccccc3)C(=O)N2[C@H]1C(=O)O
ampicillin
O=C(O)[C@@H]1CCCN1
proline
O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO
glucose
CC1(C)S[C@@H]2[C@H](NC(=O)[C@H](N)c3ccccc3)C(=O)N2[C@H]1C(=O)O
ampicillin
CC1(C)S[C@@H]2[C@H](NC(=O)[C@H](N)c3ccccc3)C(=O)N2[C@H]1C(=O)O
ampicillin
O=C(O)[C@@H]1CCCN1
proline
→
O=C(O)[C@@H]1C[C@@H](O)CN1
Hydroxyproline

反应物

CC1(C)S[C@@H]2[C@H](NC(=O)[C@H](N)c3ccccc3)C(=O)N2[C@H]1C(=O)O
ampicillin
—
3A
Cc1ncc(C[n+]2csc(CCO)c2C)c(N)n1
thiamine
—
amino acids
CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O
isopropyl-β-D-thiogalactopyranoside
CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O
IPTG
CC1(C)S[C@@H]2[C@H](NC(=O)[C@H](N)c3ccccc3)C(=O)N2[C@H]1C(=O)O
ampicillin
O=C(O)[C@@H]1CCCN1
proline
O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO
glucose
—
3B
CC1(C)S[C@@H]2[C@H](NC(=O)[C@H](N)c3ccccc3)C(=O)N2[C@H]1C(=O)O
ampicillin
CC1(C)S[C@@H]2[C@H](NC(=O)[C@H](N)c3ccccc3)C(=O)N2[C@H]1C(=O)O
ampicillin
O=C(O)[C@@H]1CCCN1
proline

试剂

无

反应条件

温度
37°CELSIUS
详细条件
See reaction.notes.procedure_details.

后处理

  1. 1
    workup.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. 2
    其他are harvested by centrifugation (5000 rpm, 5 minutes)
  3. 3
    其他the media decanted
  4. 4
    workup.ADDITIONcontaining 100 μg/mL ampicillin, 0.08M L-hydroxyproline, and 0.5M NaCl
  5. 5
    其他for 1 hour
  6. 6
    其他at 37° C.
  7. 7
    workup.ADDITION225 rpm, IPTG is added to 1 mM
  8. 8
    workup.WAITto grow for an additional 5-15 hours
  9. 9
    其他are harvested by centrifugation (5000 rpm, 10 minutes)
  10. 10
    workup.ADDITIONHydroxyproline-containing collagen
  11. 11
    其他is purified by ammonium sulfate fractionation and column chromatography

实验过程

A plasmid (pHuCol, FIG. 4) encoding the gene sequence of human Type I (α1) collagen (FIGS. 3A and 3B) (SEQ. ID. NO. 1) placed 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 NaCl. 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.

来源

DOI: 10.6084/m9.figshare.5104873.v1专利: US06927287B1uspto-grants-2005_08