A method for increasing soluble heterologous expression of plant-derived glycosyltransferase genes
A glycosyltransferase, plant-derived technology, applied in the field of increasing soluble heterologous expression of plant-derived glycosyltransferase genes, can solve the problem of low soluble expression, and achieve the effect of increasing enzyme activity and solubility
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Embodiment 1
[0033] Example 1 Construction of recombinant Escherichia coli
[0034] 1) Acquisition of the tagged protein gene
[0035] Primers were designed according to CysQ, EDA, NusA gene sequences,
[0036] CysQ upstream primers are:
[0037] 5’-gtg ccg cgc ggc agc cat atg tta gat caa gta tgc cag ctt g-3'
[0038] CysQ downstream primers are:
[0039] 5'-ctc gag tgc ggc cgc aag ctt gta aat aga cac tct gaa ccc c-3'
[0040] EDA upstream primers are:
[0041] 5’-gtg ccg cgc ggc agc cat atg aaa aac tgg aaa aca agt gca g-3'
[0042] EDA downstream primers are:
[0043] 5'-ctc gag tgc ggc cgc aag ctt cag ctt agc gcc ttc tac agc-3'
[0044] NusA upstream primers are:
[0045] 5’-gtg ccg cgc ggc agc cat atg aac aaa gaa att ttg gct gta g-3'
[0046] NusA downstream primers are:
[0047] 5'-ctc gag tgc ggc cgc aag ctt cgc ttc gtc acc gaa cca gc-3'
[0048] All primers were synthesized by Nanjing GenScript Company.
[0049] Using Escherichia coli BL21 (DE3) genome a...
Embodiment 2
[0072] Embodiment 2 Fusion expression of recombinant bacteria
[0073] Inoculate four recombinant strains containing pET-28a-UGT and pET-28a-CysQ-UGT, pET-28a-EDA-UGT and pET-28a-NusA-UGT into 5mL containing 0.05g / L kanamycin LB culture medium (0.5g / L yeast powder, 1g / L sodium chloride, 1g / L tryptone) was shaken at 37°C and 200rpm for 8 hours, and then the culture solution was inserted into 50mL LB medium according to the inoculation amount of 2%. cultured at 200rpm, 37°C for 2h, until the OD 600 When it reaches about 0.2, add 0.5mMIPTG, and then induce 16h at 30°C to collect the bacteria by centrifugation. Sonicate the bacteria, centrifuge and take the supernatant as the crude enzyme solution, and take a part for SDS-PAGE detection (results see, figure 1 ), and others were stored in a 4°C refrigerator for later use. It was found that the soluble ratio of the fusion protein CysQ-UGT was twice as high as that of UGT alone; the solubility of the fusion proteins EDA-UGT and Nus...
Embodiment 3
[0074] Example 3 Effect of temperature on fusion expression of recombinant bacteria
[0075] Inoculate two strains of recombinant bacteria containing pET-28a-UGT and pET-28a-CysQ-UGT into 5mL LB medium containing 0.05g / L kanamycin (0.5g / L yeast powder, 1g / L chloride Sodium, 1g / L tryptone), shaking culture at 37°C and 200rpm for 8h, then transfer the culture solution into a 500mL shake flask containing 50mL LB medium according to the inoculation amount of 2%, and culture at 200rpm, 37°C for 2h, wait OD 600 When it reaches about 0.2, add 0.5mM IPTG, and then induce 30h, 22h, and 16h at 20, 25, and 30°C to collect the bacteria by centrifugation. Sonicate the bacteria, centrifuge and take the supernatant as the crude enzyme solution, and take a part for SDS-PAGE detection (results see, figure 2 ), and others were stored in a 4°C refrigerator for later use. It was found that the solubility effect was the best when the induction temperature was 25°C.
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