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

Microbial Cell Factories

Technical Notes

Florian W Krainer¹  Anton Glieder¹  Helmut Schwab¹  Mudassar Ahmad¹  Thomas Vogl² 

[1] Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, 8010, Graz, Austria;Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, 8010, Graz, Austria;Queensland University of Technology, 2 George St., 4000, Brisbane, QLD, Australia;

关键词: Protein tagging;    Protein tags;    Seamless cloning;    Pichia pastoris;    Expression optimization;    Cloning strategy;    Type IIS restriction endonucleases;   

DOI  :  10.1186/s12934-015-0293-6

 received in 2015-03-31, accepted in 2015-06-30,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundTagging 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).ResultsHere 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 restriction site free cloning (RSFC) strategy presented in this paper relies on seamless cloning using type IIS restriction endonucleases. After cutting out a stuffer (placeholder) fragment from the vectors, a single PCR product can be directly inserted in frame into all 40 plasmids using blunt end or TA ligations, requiring only verification of the orientation. We have established a RSFC vector family for the commonly used protein expression host Pichia pastoris and demonstrated the system with the secretory expression of horseradish peroxidase (HRP). HRP fusions to four tags (Myc, FLAG, His, Strep) and two fusion proteins (GFP and MBP) showed a 31-fold difference in volumetric activities. C-terminal tagging caused in some cases almost a complete loss of function, whereas N-terminal tags showed moderate differences.ConclusionsThe RSFC vectors provide an unprecedented toolbox for expression optimization in P. pastoris. The results obtained with HRP underline the importance of comparing different tags to maximize activities of fusion proteins. In a similar fashion the RSFC strategy can be applied in other expression hosts to screen for optimal promoters, signal sequences or to facilitate the evaluation of (iso-) enzyme families.

【 授权许可】

CC BY   
© Vogl et al. 2015