Amino acid modified polypeptides
a technology of amino acid and polypeptide, which is applied in the field of amino acid modified polypeptides, can solve the problems of inability to produce collagen by commercial mass producers such as i>e. coli /i>, poor mechanical properties of collagen with low levels of 4-hydroxyproline, and inability to bind to a single amino acid, etc., to achieve the effect of increasing the stability of a recombinant polypeptid
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example 1
Trans-Membrane Transport
[0187] A 5 mL culture of E. coli strain DH5α (supE44 ΔlacU169 (φ801acZ ΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1) containing a plasmid conferring resistance to ampicillin (pMAL-c2, FIG. 1) was grown in Luria Broth to confluency (˜16 hours from inoculation). These cells were used to inoculate a 1 L shaker flask containing 500 mL of M9 minimal medium (M9 salts, 2% glucose, 0.01 mg / mL thiamine, 100 ∥g / mL ampicillin supplemented with all amino acids at 20 ∥g / mL) which was grown to an AU600 of 1.0 (18-20 hours). The culture was divided in half and the cells harvested by centrifugation. The cells from one culture, were-resuspended in 250 mL M9 media and those from the other in 250 mL of M9 media containing 0.5M NaCl. The cultures were equilibrated in an air shaker for 20 minutes at 37° C. (225 rpm) and divided into ten 25 mL aliquots. The cultures were returned to the shaker and 125 μl of 1M hydroxyproline in distilled H2O was added to each tube. At 2, 4, 8, 12,...
example 2
Effects of Salt Concentration on Transmembrane Transport
[0188] To determine the effects of salt concentration on transmembrane transport, an approach similar to Example 1 was taken. A S mL culture of E. coli strain DH5α (supE44 ΔlacU169 (φ80lacZ ΔM15) hsdR17 recA1 entA1 gyrA96 thi-1 relA1) containing a plasmid conferring resistance to ampicillin (pMAL-c2, FIG. 1) was grown in Luria Broth to confluency (˜16 hours from inoculation). These cells were used to inoculate a 1 L shaker flask containing 500 mL of M9 minimal medium (M9 salts, 2% glucose, 0.01 mg / nl thiamine, 100 μg / mL ampicillin supplemented with all amino acids at 20 μg / mL) that was then grown to an AU600 of 0.6. The culture was divided into three equal parts, the cells in each collected by centrifugation and resuspended in 150 mL M9 media, 150 mL M9 media containing 0.5M NaCl, and 150 mL M9 media containing 1.0M NaCl, respectively. The cultures were equilibrated for 20 minutes on a shaker at 37° C. (225 rpm) and then divid...
example 2a
Effects of Salt Concentration on Transmembrane Transport
[0189] To determine the effects of salt concentration on transmembrane transport, an approach similar to Example 1 was taken. A saturated culture of JM109 (F—) harboring plasmid pD4 (FIG. 48) growing in Luria Broth (LB) containing 100 μg / ml ampicillin (Amp) was used to inoculate 20 ml cultures of LB / Amp to an OD at 600 nm of 0.1 AU. The cultures were grown with shaking at 37° C. to an OD 600 run between 0.7 and 1.0 AU. Cells were collected by centrifugation and washed with 10 ml of M9 media. Each cell pellet was resuspended in 20 ml of M9 / Amp media supplemented with 0.5% glucose and 100 μg / ml of all of the amino acids except proline. Cultures were grown at 37° C. for 30 min. to deplete endogenous proline. After out-growth, NaCl was added to the indicated concentration, Hyp was added to 40 mM, and IPTG to 1.5 mM. After 3 hours at 37° C., cells from three 5 ml aliquots of each culture were collected separately on polycarbonate f...
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