Expression Plasmids

3.1.1. Expression Vector

The cDNA encoding for the full-length wild-type T7 RNAP has been previously cloned in the pMR-78 vector (29) and is presented in Fig. 1. The expression of the insert is under the control of the Ptac promoter, which is inducible by IPTG. This vector has a specific bla gene that confers ampicillin resistance for colony selection and growth. It is designed to express an N-terminal his-tagged T7 RNAP for purification with a Ni2+/iminodiacetic acid affinity column.

3.1.2. cDNA Constructs

The cDNAs used should contain a T7 promoter at the 5' end of the gene of interest. For protein production in oocytes, the cDNA should encode a Kozak's sequence for initiation of translation (30) and a stop codon. cRNA stability, and thus protein yield, can be greatly improved by the presence of a poly A tail sequence at the 3' end of the gene of interest (31).

0 0

Post a comment