Table 1.
RSFC vector family designed for P. pastoris
| # | Name | Tag/Fusion protein, positiona and lengthb | TEV protease cleavage site | Mode of expression | EAEA repeat | Selection markerc | Plasmid size (bp) | ||
|---|---|---|---|---|---|---|---|---|---|
| 1 | PPpRSFC | – | – | – | NA | Intracellular | NA | Zeocin | 4,840 |
| 2 | pPpRSFC-MFalpha | – | – | – | NA | Secretory | Yes | Zeocin | 5,104 |
| 3 | pPpRSFC-MFalpha-noEAEA | – | – | – | NA | Secretory | No | Zeocin | 5,092 |
| 4 | pPpRSFC-pGAP | – | – | – | NA | Intracellular | NA | Zeocin | 3,771 |
| 5 | pPpRSFC-pGAP-MFalpha | – | – | – | NA | Secretory | Yes | Zeocin | 4,035 |
| 6 | pPpRSFC-pGAP-MFalpha-noEAEA | – | – | – | NA | Secretory | No | Zeocin | 4,023 |
| 7 | pPpRSFC-N-eGFP | eGFP | N | 240 | No | Intracellular | NA | Zeocin | 5,584 |
| 8 | pPpRSFC-C-eGFP | eGFP | C | 239 | No | Intracellular | NA | Zeocin | 5,584 |
| 9 | pPpRSFC-MFalpha-N-eGFP | eGFP | N | 239 | No | Secretory | Yes | Zeocin | 5,848 |
| 10 | pPpRSFC-MFalpha-C-eGFP | eGFP | C | 239 | No | Secretory | Yes | Zeocin | 5,848 |
| 11 | pPpRSFC-N-Myc | MYC | N | 11 | No | Intracellular | NA | Zeocin | 4,870 |
| 12 | pPpRSFC-C-Myc | MYC | C | 10 | No | Intracellular | NA | Zeocin | 4,870 |
| 13 | pPpRSFC-MFalpha-N-Myc | MYC | N | 10 | No | Secretory | Yes | Zeocin | 5,134 |
| 14 | pPpRSFC-MFalpha-C-Myc | MYC | C | 10 | No | Secretory | Yes | Zeocin | 5,134 |
| 15 | pPpRSFC-N-FLAG | FLAG | N | 9 | No | Intracellular | NA | Zeocin | 4,864 |
| 16 | pPpRSFC-C-FLAG | FLAG | C | 8 | No | Intracellular | NA | Zeocin | 4,864 |
| 17 | pPpRSFC-MFalpha-N-FLAG | FLAG | N | 8 | No | Secretory | Yes | Zeocin | 5,128 |
| 18 | pPpRSFC-MFalpha-C-FLAG | FLAG | C | 8 | No | Secretory | Yes | Zeocin | 5,128 |
| 19 | pPpRSFC-N-His-ncs | His | N | 7 | No | Intracellular | NA | Zeocin | 4,858 |
| 20 | pPpRSFC-C-His-ncs | His | C | 6 | No | Intracellular | NA | Zeocin | 4,858 |
| 21 | pPpRSFC-MFalpha-N-His-ncs | His | N | 6 | No | Secretory | Yes | Zeocin | 5,122 |
| 22 | pPpRSFC-MFalpha-C-His-ncs | His | C | 6 | No | Secretory | Yes | Zeocin | 5,122 |
| 23 | pPpRSFC-N-His | His | N | 7 | Yes | Intracellular | NA | Zeocin | 4,879 |
| 24 | pPpRSFC-C-His | His | C | 6 | Yes | Intracellular | NA | Zeocin | 4,879 |
| 25 | pPpRSFC-MFalpha-N-His | His | N | 6 | Yes | Secretory | Yes | Zeocin | 5,143 |
| 26 | pPpRSFC-MFalpha-C-His | His | C | 6 | Yes | Secretory | Yes | Zeocin | 5,143 |
| 27 | pPpRSFC-N-MBP | MBP | N | 367 | Yes | Intracellular | NA | Zeocin | 5,959 |
| 28 | pPpRSFC-C-MBP | MBP | C | 366 | Yes | Intracellular | NA | Zeocin | 5,959 |
| 29 | pPpRSFC-MFalpha-N-MBP | MBP | N | 366 | Yes | Secretory | Yes | Zeocin | 6,223 |
| 30 | pPpRSFC-MFalpha-C-MBP | MBP | C | 366 | Yes | Secretory | Yes | Zeocin | 6,223 |
| 31 | pPpRSFC-N-Strep | Strep | N | 9 | Yes | Intracellular | NA | Zeocin | 4,885 |
| 32 | pPpRSFC-C-Strep | Strep | C | 8 | Yes | Intracellular | NA | Zeocin | 4,885 |
| 33 | pPpRSFC-MFalpha-N-Strep | Strep | N | 8 | Yes | Secretory | Yes | Zeocin | 5,149 |
| 34 | pPpRSFC-MFalpha-C-Strep | Strep | C | 8 | Yes | Secretory | Yes | Zeocin | 5,149 |
| 35 | pPpRSFC-HIS | – | – | – | NA | Intracellular | NA | HIS4 | 7,683 |
| 36 | pPpRSFC-HIS-MFalpha | – | – | – | NA | Secretory | Yes | HIS4 | 7,947 |
| 37 | pPpRSFC-HIS-MFalpha-noEAEA | – | – | – | NA | Secretory | No | HIS4 | 7,935 |
| 38 | pPpRSFC-HIS-pGAP | – | – | – | NA | Intracellular | NA | HIS4 | 6,614 |
| 39 | pPpRSFC-HIS-pGAP-MFalpha | – | – | – | NA | Secretory | Yes | HIS4 | 6,878 |
| 40 | pPpRSFC-HIS-pGAP-MFalpha-noEAEA | – | – | – | NA | Secretory | No | HIS4 | 6,866 |
NA not applicable, ncs no TEV protease cleavage site.
aN- or C- terminal fusion to the POI.
bLength in amino acids (intracellular N- terminal tags are because of the start codon one aa longer, the TEV protease cleavage site (seven aa) is not included in this number).
cZeocin selection is applicable for E. coli and P. pastoris, HIS4 plasmids are based on ampicillin selection in E. coli and used in combination with a histidine auxotrophic (his4) P. pastoris strain.