反应 #486559

反应方程式

ampicillin

proline

IPTG

proline

ampicillin

ampicillin

thiamine

isopropyl-β-D-thiogalactopyranoside

glucose

ampicillin

→

Hydroxyproline

反应条件

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 NaC1
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.ADDITIONThe hydroxyproline-containing collagen fragment
11. 11
    其他is purified by ammonium sulfate fractionation and column chromatography

实验过程

A plasmid (pHuCol-Fl, FIG. 6) encoding the gene sequence of the first 80 amino acids of human Type 1 (α1) collagen (FIG. 5 (Seq. ID No. 2) 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 NaC1. 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. The hydroxyproline-containing collagen fragment is purified by ammonium sulfate fractionation and column chromatography.

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