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Expression system

A technology of expression system and expression box, applied in the field of expression system, can solve problems such as undesirable and time-consuming

Active Publication Date: 2012-09-26
FUJIFILM DIOSYNTH BIOTECH UK
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This is time consuming and undesirable

Method used

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Examples

Experimental program
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Effect test

Embodiment 3

[0193] Remove the CLD032 vial from the -80°C freezer and allow it to thaw. Inoculate 10 μl of the thawed glycerol stock solution into 5 ml of LB culture medium supplemented with tetracycline (10 μg / ml) and glucose (1 g / L) (LB, 5 g / L yeast extract (Oxoid), 10 g / L tryptone (Oxoid) and 5g / L sodium chloride). Incubate for 16 hours at 37°C in an orbital shaker. 500 μl of this culture was then used to inoculate two 250 ml Erlenmeyer flasks containing 50 ml of LB broth (composition as described above). The Erlenmeyer flasks were incubated in an orbital shaker at 37°C, 200 rpm. Monitor growth until OD 600 = 0.5-0.7. At this time, one Erlenmeyer flask was induced with IPTG (isopropyl-β-D-1-thiogalactopyranoside) at a final concentration of 0.05 mM, while the second Erlenmeyer flask was not induced and kept for monitoring basal expression. Incubation was continued under the conditions described above, during which time samples were taken for growth measurements, hTNF[alpha] accumul...

Embodiment 4

[0199] Remove the CLD018 vials from the -80 °C freezer and allow them to thaw. Inoculate 10 μl of the thawed glycerol stock solution into 5 ml of LB culture medium supplemented with tetracycline (10 μg / ml) and glucose (1 g / L) (LB, 5 g / L yeast extract (Oxoid), 10 g / L tryptone (Oxoid) and 5g / L sodium chloride). The seed cultures were incubated for 16 hours at 37°C in an orbital shaker. 500 [mu]l of seed culture was then used to inoculate a 250 ml Erlenmeyer flask containing 50 ml of LB broth (composition as described above). The Erlenmeyer flasks were incubated in an orbital shaker at 37°C, 200 rpm. Monitor growth until OD 600 = 0.5-0.7. At this point the Erlenmeyer flasks were induced with IPTG (isopropyl-β-D-1-thiogalactopyranoside) at final concentrations of 0.05 mM and 1 mM. The Erlenmeyer flask was also left without induction, and the incubation of the Erlenmeyer flask was continued under the above conditions, during which time samples were taken for growth measurement...

Embodiment 5

[0204] Remove the CLD026 vials from the -80 °C freezer and allow them to thaw. Inoculate 10 μl of the thawed glycerol stock solution into 5 ml of LB culture medium supplemented with tetracycline (10 μg / ml) and glucose (1 g / L) (LB, 5 g / L yeast extract (Oxoid), 10 g / L tryptone (Oxoid) and 5g / L sodium chloride). Incubate for 16 hours at 37°C in an orbital shaker. 500 [mu]l of this culture was then used to inoculate a 250 ml Erlenmeyer flask containing 50 ml of LB broth (composition as described above). The Erlenmeyer flasks were incubated in an orbital shaker at 37°C, 200 rpm. Monitor growth until OD 600 = 0.5-0.7. At this point the Erlenmeyer flasks were induced with IPTG (isopropyl-β-D-1-thiogalactopyranoside) at final concentrations of 0.05 mM and 0.005 mM. The Erlenmeyer flask was also left without induction, and the incubation was continued under the above-mentioned conditions, during which samples were taken for growth measurement and hTNFα accumulation in bacterial ce...

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Abstract

A perfect palindrome operator sequence-based protein expression system is provided. The expression system comprises a promoter; and a perfect palindrome operator sequence, wherein the promoter is not T7. The expression system is preferably employed for the production of recombinant proteins by fermentation.

Description

[0001] This application is a divisional application of the Chinese patent application 200780011276.8 "Expression System" with a filing date of February 1, 2007. technical field [0002] The present invention relates to expression systems suitable for microbial expression of recombinant polypeptides. Background technique [0003] A T7-based protein expression system based on a complete palindromic operator sequence is known from US Pat. No. 6,537,779. T7-based systems have disadvantages because the operation of the T7 system requires phage polymerase, which is usually provided by inserting a λDE3 prophage expressing the desired phage polymerase into an E. coli host strain to generate a lysogenic host strain. Phage polymerase can also be delivered into cells by infection with a specific lambda transducing phage carrying a gene for a phage polymerase (eg, T7 RNA polymerase). The lambda DE3 prophage lacks the genetic elements required for excision of the prophage to form lytic ...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N1/21C12N1/19C12N5/10C12N15/70C12N15/81C12N15/85C12P21/00
CPCC12N15/72C12N15/73C12N15/78C12N15/70C12N15/81C12N15/85C12N15/09C12N1/205C12R2001/19C12P21/00
Inventor I·J·霍奇森C·D·J·伦农B·V·卡拉
Owner FUJIFILM DIOSYNTH BIOTECH UK
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