title: 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 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.
  
  
  CONCLUSIONS
  The 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.
authors.affiliation: Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, 8010, Graz, Austria. thomas.vogl@aon.at.|Queensland University of Technology, 2 George St., Brisbane, QLD, 4000, Australia. thomas.vogl@aon.at.
authors.forename: Thomas
authors.initials: T
authors.lastname: Vogl
authors.affiliation: Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, 8010, Graz, Austria. mudassar094@hotmail.com.
authors.forename: Mudassar
authors.initials: M
authors.lastname: Ahmad
authors.affiliation: Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, 8010, Graz, Austria. florian.krainer@tugraz.at.
authors.forename: Florian W
authors.initials: FW
authors.lastname: Krainer
authors.affiliation: Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, 8010, Graz, Austria. helmut.schwab@tugraz.at.
authors.forename: Helmut
authors.initials: H
authors.lastname: Schwab
authors.affiliation: Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, 8010, Graz, Austria. a.glieder@tugraz.at.
authors.forename: Anton
authors.initials: A
authors.lastname: Glieder
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins