Method for expressing and purifying novel cell reprogramming factor
A technology of cells and expression vectors, applied in the field of expression and purification of new cell reprogramming factors, which can solve the problems of reduced transmembrane activity
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Embodiment 1
[0026] Example 1 Construction of pET-HisSu-TN-Oct4 fusion protein expression vector
[0027] use as figure 1 The indicated expression vector pET-3c (purchased from Novagen, USA) clones and expresses the HisSu-TN-Oct4 fusion protein of the present invention.
[0028] The pET-3c vector has multiple cloning sites. According to the restriction site of pET-3c vector and the cDNA sequence of Oct4 protein, the following primers were designed:
[0029] OctF:GC GGTACC GCTGGACACCTGGCTTCAG (SEQ ID NO.3); contains Kpn I restriction site (underlined).
[0030] OctR:GA AGATCT TCAGTTTGAATGCATGGGAGAGCCC (SEQ ID NO. 4), containing a Bgl II restriction site (underlined).
[0031] Oct4 cDNA was amplified using TetO-FUW-OSKM plasmid as a template. The PCR reaction conditions were: 1×PCR reaction buffer, 0.5 μM Oct-F, 0.5 μM Oct-R, 1 μg TetO-FUW-OSKM plasmid, 2U DNA polymerase (Fermentas), 50 μM dATP, 50 μM dTTP, 50 μM dCTP, 50μM dGTP, 1.5mM MgCl2, adjust the reaction volume to 50μl with ...
Embodiment 2
[0036] Example 2 Expression and purification of pET-HisSu-TN-Oct4 expression vector
[0037] The recombinant DNA plasmid pET-HisSu-TN-Oct4 obtained above was transformed into Escherichia coli Rossate (DE3) (purchased from Novagen, USA). The transformants of Escherichia coli Rossate (DE3) containing pET-HisSu-TN-Oct4 were inoculated in LB medium containing 50 μg / ml of ampicillin and chloramphenicol, and cultured overnight at 37°C. Inoculate into LB liquid medium (containing ampicillin and chloramphenicol) with 1% inoculum amount, and culture on a shaker at 37°C until OD 600 0.5 to 0.6. Human isopropyl-β-D-thiogalactopyranoside (IPTG for short) was added to the culture solution to a final concentration of 0.5 mM, induced at 30°C for 4 hours, and the cells were collected by centrifugation. The bacteria were resuspended in 50 mM sodium phosphate buffer (containing 300 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF for short), pH 7.4). Sonicate the cell wall, centrifuge at 10...
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