Restriction site free cloning (RSFC) plasmid family for seamless, sequence independent cloning in Pichia pastoris.

ABSTRACT:

Background

Tagging proteins is a standard method facilitating protein detection, purification or targeting. When tagging a certain protein of interest, it is challenging to predict which tag will give optimal results and will not interfere with protein folding, activity or yields. Ideally, multiple tags and positions are tested which however complicates molecular cloning and expression vector generation. In conventional cloning, tags are either added on PCR primers (requiring a distinct primer and PCR product per tag) or provided on the vector (typically leaving a restriction site scar).

Results

Here we report a vector family of 40 plasmids allowing simple, seamless fusions of a single PCR product with various N- and C-terminal tags, signal sequences and promoters. The restr

SUBMITTER: Vogl T 

PROVIDER: S-EPMC4501187 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature