A cell-permeable Tat expression vector was prepared as described in a previous study [27 (link)]. Sense primer 5′-CTCGAGATGCGCCTCCGC-3′ and antisense primer 5′-GGATCCTTAGAGATCCTCCTGTGCC-3′ were used to amplify cDNA for CRIP1a by PCR. The PCR product was subcloned in a TA cloning vector (pGEM®-T easy vector; Promega Corporation, Madison, WI, USA) and ligated into the Tat expression vector to produce a Tat-His-CRIP1a fusion protein. The Tat domain consists of 9 amino acids, RKKRRQRRR, and is connected with a 6xHistidine tag. They are inserted in the N-terminal of CRIP1a. A His-CRIP1a (Control-CRIP1a) without the Tat domain was also prepared to use as a control.
The Tat-His-CRIP1a and His-CRIP1a-containing plasmids were transformed into Escherichia coli BL21 cells to produce both proteins. Isopropyl-β-d-thiogalactoside (0.1 mM, Duchefa, Haarlem, The Netherlands) was given to the bacterial cells at 18 °C for 8 h, and the harvested cells were purified with a Ni2+-nitrilotriacetic acid Sepharose affinity column and PD-10 column chromatography (Amersham, Braunschweig, Germany). To estimate the concentration of purified proteins, a Bradford assay was performed.
The Tat-His-CRIP1a and His-CRIP1a-containing plasmids were transformed into Escherichia coli BL21 cells to produce both proteins. Isopropyl-β-d-thiogalactoside (0.1 mM, Duchefa, Haarlem, The Netherlands) was given to the bacterial cells at 18 °C for 8 h, and the harvested cells were purified with a Ni2+-nitrilotriacetic acid Sepharose affinity column and PD-10 column chromatography (Amersham, Braunschweig, Germany). To estimate the concentration of purified proteins, a Bradford assay was performed.
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