pBGP3-G1NkBG

Protein Expression Eukaryotic cells - P. pastoris cellulases

Experiment
Protein Expression Eukaryotic cells - P. pastoris cellulases
Product
pBGP3-G1NkBG from Manabu Arioka, Department of Biotechnology, The University of To
Manufacturer
Manabu Arioka, Department of Biotechnology, The University of To

Protocol tips

Protocol tips
The resulting linear DNA fragment was self-ligated, giving rise to pBGP3 (Fig. 1A and B). The DNA sequences of all the plasmids were checked.

Publication protocol

Briefly, a sequence encodingmyc-epitope and six consecutive histidines (6His) of pBGP1 was amplified by PCR using Pyrobest DNA polymerase (Takara, Japan) and a set of forward (5 0 -CAATTGGAACAAAAACTCATC TCAGAAGAGGATCG-3 0 ) and reverse (5 0 -GAATTC- ATGATGATGATGATGATGGTCGACGG-3 0 ) primers containingMunI andEcoR I restriction sites respec- tively (underlined) at the 5 0 -ends. The amplified DNA fragment was phosphorylated and inserted into the EcoR V site of pBluescript II SK(รพ). This plasmid, pBS-BGP2, was digested withMunI andEcoR I and inserted into theEcoR I site of pBGP1, generating pBGP2. Since only the use of N-terminal tags was intended, the presence of C-terminal tags in pBGP2 was undesired and useless. To remove the repetitive C-terminal tag sequence from pBGP2, a pair of back-to- back, oppositely directed primers (5 0 -GTTTTAGCCTT- AGACATGACTGTTCCTCA-3 0 and 5 0 -TTCTAGAAA- GCTGGCGGCCG-3 0 ) was used to amplify the entire sequence of pBGP2, except for the region coding for the redundant C-terminal tags. The resulting linear DNA fragment was self-ligated, giving rise to pBGP3 (Fig. 1A and B). The DNA sequences of all the plasmids were checked.

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