Ampicillin → L-Hydroxyproline

Temperature

37 °C

A plasmid (pGST-CM4, FIG. 60) containing the gene for collagen mimetic 4 (CM4, FIG. 61) (SEQ. ID. NO. 39) genetically linked to the 3′ end of the gene for S. japonicum glutathione S-transferase was used to transform by electroporation proline auxotrophic E. coli strain JM109 (F-). Transformation cultures were plated on LB agar containing 100 μg/ml ampicillin. After overnight incubation at 37° C., a single colony from a fresh transformation plate was used to inoculate 5 ml of LB media containing 100 μg/ml ampicillin. After overnight growth at 37° C., 500 μl of this culture was centrifuged, the supernatent discarded, and the cell pellet washed once with 500 μl of M9 medium (1× M9 salts, 0.5% glucose, 1 mM MgCl2, 0.01% thiamine, 200 μg/ml glycine, 200 μg/ml alanine, 100 μg/ml of the other amino acids except proline, and 400 μg/ml ampicillin). The cells were finally suspended in 5 ml of M9 medium containing 10 μm/ml proline and 2 ml of this was used to inoculate 30 ml of M9 medium containing 10 μg/ml proline. After incubation with shaking at 37° C. for 8 hours, the culture was centrifuged and the cell pellet washed once with M9 medium containing 5 μg/ml proline. The pellet was resuspended in 15 ml of M9 medium containing 5 μg/ml of proline and this culture was used to inoculate 1 L of M9 medium containing 5 μg/ml of proline. This culture was grown for 18 hours at 37° C. to proline starvation. At this time, the culture was centrifuged, the cells washed once with M9 medium (with no proline), and the cells resuspended in 1 L of M9 medium containing 80 mM hydroxyproline, 0.5M NaCl, and 1.5 mM isopropyl-β-D-thiogalactopyranoside. Incubation was continued at 37° C. with shaking for 22 hours. The cultures were centrifuged and the cell pellets stored at −20° C. until processed further.

1. additionA plasmid (pGST-CM4, FIG. 60) containing the gene for collagen mimetic 4 (CM4, FIG. 61) (SEQ
2. waitAfter overnight growth at 37° C.
3. washthe cell pellet washed once with 500 μl of M9 medium (1× M9 salts, 0.5% glucose, 1 mM MgCl2, 0.01% thiamine, 200 μg/ml glycine, 200 μg/ml alanine, 100 μg/ml of the other amino acids except proline, and 400 μg/ml ampicillin)
4. additioncontaining 10 μm/ml proline and 2 ml of
5. additioncontaining 10 μg/ml proline
6. washthe cell pellet washed once with M9 medium
7. additioncontaining 5 μg/ml proline
8. additioncontaining 5 μg/ml of proline
9. additioncontaining 5 μg/ml of proline
10. waitThis culture was grown for 18 hours at 37° C. to proline starvation
11. washthe cells washed once with M9 medium (with no proline), and the cells
12. additioncontaining 80 mM hydroxyproline, 0.5M NaCl, and 1.5 mM isopropyl-β-D-thiogalactopyranoside
13. customwas continued at 37° C.
14. stirringwith shaking for 22 hours
15. customstored at −20° C.

ORD record

ord-9571771268504cf7b7653dc95934c910

Source dataset

uspto-grants-2005_08

DOI

10.6084/m9.figshare.5104873.v1

Patent number

US06927287B1