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

Abstract

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0293-6) contains supplementary material, which is available to authorized users.

Effect of Plasmid Design and Type of Integration Event on Recombinant Protein Expression in Pichia pastoris

“…Notably, we obtained higher specific integration rates in the GUT1 locus by homologous recombination than those reported in previous studies (12), despite using identical 5= and 3= homologous regions and expression cassettes based on a similar vector family. Although hard to rationalize, this difference may be related to the insertion of longer sequences (P AOX1 , eGFP, and terminator expression cassette) between the GUT1 5= and 3= regions in our study, as the previous knockout cassette contained only a resistance marker (12,31). Elucidating the exact mechanisms will require further studies.…”

“…Thus, we set up experiments mimicking commonly used P. pastoris protocols with regard to expression plasmids, how the plasmids are linearized to target integration, and amounts of DNA used. The most commonly used P. pastoris plasmids (11,31,32) are propagated in E. coli in circular form and linearized prior to transformation (as free DNA ends strongly increase the rate of integration into the genome [33][34][35][36][37]). As most standard plasmids contain the strong, methanol-inducible AOX1 promoter (P AOX1 ), enzymes cutting at the 5= end of P AOX1 (typically BglII) or within the AOX1 promoter (commonly SacI) are used.…”

Integration event induced changes in recombinant protein productivity in Pichia pastoris discovered by whole genome sequencing and derived vector optimization

“…The absence of false-positive clones strongly suggests that the integration of the marker cassette found in JPS056 and JPS060 was indeed mediated by a double crossing over event using the AOX1 TT and 3′ AOX1 HS regions. Multiple commercial and non-commercial vectors for P. pastoris are targeted for AOX1 replacement, while putting the gene of interest under the control of p AOX1 and AOX1 TT [ 49 , 50 ]. Integration of only the marker cassette by the mechanism described here can potentially also occur using these plasmids.…”

Non-canonical integration events in Pichia pastoris encountered during standard transformation analysed with genome sequencing

“…Using blunt-end or rare cutters for plasmid linearization should also impede inadvertent co-integrations in these situations. Newly developed plasmids were recently published that offer sites for blunt-end linearization of an expression cassette targeting the AOX1 locus for replacement56. While evolutionary horizontal gene transfer from bacteria to yeast (e.g.…”