Root > Products > Strep-Tactin® & Strep-tag® > Expression Vectors > Mammalia > Cloning Procedure for High-level of Protein Expression in Mammalian Cells Using pEXPR-IBA Vectors.

Technical information

 
Cloning system

Cloning procedure (Classic Cloning)

The polylinkers of the expression vectors carry the restriction sites BsaI (isoschizomer Eco31I) and BsmFI (New England Biolabs, MBI Fermentas) which allow the precise fusion of the structural gene with the vector-encoded functional elements (including Strep-tag® II, 6xHistidine-tag and, depending on the vector, OmpA-signal sequence, start codon, protease cleavage site or stop codon. This is easily achieved by adapting both ends of the coding region of the structural gene via PCR.
The cloning strategy is described for pASK-IBA3 (see cloning scheme). If a different vector is to be used, the cloning strategy has to be adapted accordingly. The essential primer sequences for each vector are described here or may be deduced by using the Primer D'Signer software (see below).
In cases where other restriction sites are intended to be used for cloning, care must be taken to ensure the in-frame fusion of the structural gene and the vector encoded functional elements.
In the vectors pASK-IBA4 to pASK-IBA7, pASK-IBA35, pASK-IBA37, pASK-IBA44 and pASK-IBA45 with N-terminal affinity tags (see vector overview) the tag is followed by the linker sequence 5'-GGCGCC-3', which is recognized by four different restriction enzymes (KasI, NarI, EheI and BbeI). These four enzymes cut the linker sequence in four different ways. Thus, cleavage with the suitable enzyme and a subsequent filling reaction enable the production of blunt ends in all reading frames in case the target gene insert requires a particular reading frame.
To avoid the incorporation of base substitutions, PCR should be performed with a proof-reading DNA polymerase such as Pfu (Stratagene). 3' phosphorothioate-protected primers should be used in order to avoid 3'5' degradation by the proof-reading activity.

Eco31I and BsaI belong to the Type IIS restriction enzymes which cleave the DNA double strand outside their recognition site. Thereby, the digestion with one single enzyme can generate two different independent sticky ends with 4-base 5'-overhangs allowing directional cloning. In addition, the digestion reaction removes the recognition sequence not affecting the encoded amino acid sequence and expressing authentic protein.

 

Cloning scheme (demonstrated for pASK-IBA3)

* If a different vector is to be used, the cloning strategy has to be adapted accordingly. In principle the procedure applies to pASK-IBA; pPR-IBA and pEXPR-IBA vectors.

Precise fusion using Eco31I or BsaI

 

Vector features

pEXPR-IBA mammalian expression vectors

MCS and vector map of pEXPR-IBA mammalian expression vectors

Sequencing primers

Product
amount
cat. no.
Forward sequencing primer for pEXPR-IBA vectors
1 nmol, 10 pmol/µl
HPLC purified
5-0000-121
Reverse sequencing primer for pEXPR-IBA vectors
1 nmol, 10 pmol/µl
HPLC purified
5-0000-122
Forward and reverse sequencing primers for pEXPR-IBA vectors
1 nmol each, 10 pmol/µl HPLC purified
5-0000-124