文献类型：专利

英文题名：Heterogenous soluble expression of exogenous gene in Escherichia coli by transferring recombinant exogenous protein expression carrier and carrier for expressing molecular chaperone GroEL/GroES common to Escherichia coli strain

作者：FAN Y;HUA Q;LI T;LIU N;MA N;WEI L;ZHANG J;ZHOU Y

机构：[1]UNIV EAST CHINA SCI&TECHNOLOGY

申请号：CN102505023-A

申请日：2011-11-18

公开日：2012-06-20

语种：英文

收录：DERWENT

摘要：NOVELTY - Heterogenous soluble expression of exogenous gene in Escherichia coli involves structuring a recombinant exogenous protein expression carrier for expressing recombinant exogenous gene; transferring the recombinant exogenous protein expression and a carrier for expressing molecular chaperone GroEL/GroES common to Escherichia coli strain to obtain soluble foreign proteins gene expression of engineered Escherichia coli. USE - Method for heterogeneous soluble expression of exogenous gene in Escherichia coli (claimed). DETAILED DESCRIPTION - INDEPENDENT CLAIMS are included for: (1) an exogenous protein comprising gene of 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase encoded in Actinosynnema pretiosum; (2) an E. coli gene expression engineered microorganism containing carrier for expressing molecular chaperone GroEL/GroES carrier and recombinant exogenous protein expression; (3) preparation of DAHP synthase encoded in Actinosynnema pretiosum, comprising culturing E. coli gene expression engineered microorganism and obtaining the DAHP synthase from the culture; (4) purification of recombinant DAHP synthase expressed by E. coli gene expression engineered microorganism, comprising washing the E. coli gene expression engineered microorganism with phosphate buffered saline (PBS) buffer liquid, adding combined buffer liquid, performing ultrasonic treatment for 99 times per 15 ml for 2 seconds at 200 W, and then stopping for 9 seconds, centrifuging obtained thallus for 25 minutes at 12000 revolutions/minute and at 4 degrees C, taking the supernatant and adding it to 10 mM iminazole, sampling the mixed liquid, washing it with buffer liquid containing 20 mM iminazole, and collecting the eluted target protein using elution buffer liquid containing 200 mM iminazole, performing ultra-filtering, centrifuging and concentrating the collected eluent by an ultra-filter centrifuging tube; and (5) detection of enzymatic activity of DAHP synthase, comprising preparing 75 mu L of reaction system comprising 1.5 mM phosphoenolpyruvate (7.5 mu L) and 1.5 mM erythrose-4-phosphate (1.5 mu L), and DAHP synthase liquid, adding 100 mM manganese sulfate, and supplying rest of Tris-hydrochloride (ph=7.5), placing the reaction system at 30 degrees C and reacting for 10 minutes, adding of 40% (wt./vol.) of trichloroacetic acid (100 mu L) and ending the enzymatic reaction, then adding 100 mM potassium periodate (25 mu L) into the mixed fluid and reacting for 30 minutes at 37 degrees C, then adding 18% (wt./vol.) of sodium sulfite (25 mu L) NaSO3 in the mixed fluid to remove excess oxidant and adding 2.16% (wt./vol.) of thiobarbituric acid (175 mu L), metal-bathing and boiling for 10 minutes at 100 degrees C, and adding the mixed fluid to a pipe and cooling to room temperature, after centrifuging, taking supernatant (200 mu L) in a trace cuvette, and reading light absorption value at 549 nm in a UV-1800 spectrophotometer.