Reaktion #671950

Reaktionsgleichung

ampicillin

proline

IPTG

proline

ampicillin

ampicillin

thiamine

isopropyl-β-D-thiogalactopyranoside

glucose

ampicillin

→

Hydroxyproline

Reaktionsbedingungen

Aufarbeitung

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
   Sonstigeare harvested by centrifugation (5000 rpm, 5 minutes)
3. 3
   Sonstigethe media decanted
4. 4
   workup.ADDITIONcontaining 100 μg/mL ampicillin, 0.08M L-hydroxyproline, and 0.5M NaCl
5. 5
   Sonstigefor 1 hour
6. 6
   Sonstigeat 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
   Sonstigeare harvested by centrifugation (5000 rpm, 10 minutes)
10. 10
    workup.ADDITIONThe hydroxyproline-containing collagen fragment
11. 11
    Sonstigeis purified by ammonium sulfate fractionation and column chromatography

Vorschrift

A plasmid (pHuCol-FI, 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 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. The hydroxyproline-containing collagen fragment is purified by ammonium sulfate fractionation and column chromatography.

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