Expression vectors, hosts and protocols
A custom Gateway® destination vector was built for each recombinant host, so a catalytic-domain insert in pDONR221 can be moved into any host with the appropriate N- or C-terminal fusion by a single LR reaction. Every vector below is available as an annotated GenBank file, a map and a full sequence PDF.
Mammalian expression vectors
N-terminal fusions
| Vector | Backbone | Appended sequences and tags | Used in | Files |
|---|---|---|---|---|
| pGEn1-DEST | pXKM | N-terminal signal sequence, His tag, StrepII tag | 339 records | |
| pGEn2-DEST | pXKM | N-terminal signal sequence, His tag, AviTag, superfolder GFP | 339 records | |
| pGEn3-DEST | pXKM | N-terminal signal sequence, AviTag, superfolder GFP, Fc fragment | 339 records |
C-terminal fusions
| Vector | Backbone | Appended sequences and tags | Used in | Files |
|---|---|---|---|---|
| pGEc1-DEST | pXKM | C-terminal His tag, StrepII tag, termination codon | 339 records | |
| pGEc2-DEST | pXKM | C-terminal superfolder GFP, AviTag, His tag, termination codon | 339 records |
Baculovirus expression vectors
Polyhedrin-driven destination vectors
| Vector | Backbone | Appended sequences and tags | Used in | Files |
|---|---|---|---|---|
| Ac-polh-CtermHisStrep-DEST | AcMNPV polyhedrin locus | C-terminal His tag, StrepII tag | 339 records | |
| Ac-polh-NtermMelHisStrep-DEST | AcMNPV polyhedrin locus | N-terminal melittin signal sequence, His tag, StrepII tag | 339 records | |
| Ac-polh-NtermMelHisAviTagGFP-DEST | AcMNPV polyhedrin locus | N-terminal melittin signal sequence, 8xHis, AviTag, superfolder GFP | — |
Bacterial expression vectors
N-terminal fusions
| Vector | Backbone | Appended sequences and tags | Used in | Files |
|---|---|---|---|---|
| pET16-DEST | pET16 | N-terminal His tag | 58 records | |
| pET32-DEST | pET32 | N-terminal thioredoxin (Trx) fusion, His tag | 58 records |
All constructs carry a TEV protease cleavage site between the insert and the appended tags, so the tag cassette can be removed after affinity purification. Insertion is at the Gateway® recombination site in every case.
Host cells and strains
| System | Cell line / strain | Representative source |
|---|---|---|
| Bacterial | BL21(DE3)pLysS | Promega, cat. no. L1191 |
| Bacterial | ArcticExpress (DE3) | Agilent Technologies, cat. no. 203192 |
| Bacterial | Tuner™(DE3)pLysS | EMD4Biosciences, cat. no. 70624 |
| Insect | Sf9 | ATCC (CRL-1711); Invitrogen (B82501); EMD (71023, TriEx™ Sf9) |
| Insect | Sf21 | Invitrogen (B82101); Clontech (631411) |
| Insect | High Five™ (BTI Tn 5B1-4), from Trichoplusia ni | Invitrogen (B85502) |
| Mammalian | FreeStyle™ 293-F | Life Technologies, cat. no. R790-07 |
| Mammalian | HEK293S GnTI− | ATCC, cat. no. CRL-3022 |
Most baculovirus expression testing in this project used Sf9 as the host. Sf9 is a lepidopteran line clonally derived from IPLB-SF21-AE ("Sf21"), itself isolated from ovaries of Spodoptera frugiperda (fall armyworm) pupae. Other lepidopteran lines can also serve as hosts.
Media, reagents and additives
| System | Medium / reagent | Source |
|---|---|---|
| Insect | TNM-FH (Hink's TNM-FH) | Sigma Aldrich; BD Biosciences; HyClone (Thermo Scientific) |
| Insect | ESF921 (serum-free) | Expression Systems |
| Insect | Sf900 and Express Five (serum-free) | Invitrogen |
| Insect | EX-CELL (serum-free) | Sigma Aldrich |
| Insect | SFX and SFM4 (serum-free) | HyClone (Thermo Scientific) |
| Mammalian | FreeStyle™ 293 Expression Medium | Life Technologies, cat. no. 12338026 |
| Mammalian | Ex-Cell® 293 serum-free medium | Sigma, cat. no. 14571C |
| Mammalian | ESF serum-free medium | Expression Systems, cat. no. 98-001 |
| Mammalian | Polyethylenimine (PEI), transfection reagent | Polysciences, cat. no. 23966 |
| Mammalian | Valproic acid (VPA), expression enhancer | Sigma, cat. no. P4543 |
Protocols
Further reading on insect cell culture
- Summers, M.D. and Smith, G.E. (1987) A manual of methods for baculovirus vectors and insect cell culture procedures. Texas Agricultural Experiment Station Bulletin #1555.
- O'Reilly, D.R., Miller, L.K. and Luckow, V.A. (1992) Baculovirus Expression Vectors. W.H. Freeman and Company, New York.
- Murhammer, D.W. (ed.) (2007) Baculovirus and insect cell expression protocols, 2nd edn. Methods in Molecular Biology vol. 388. Humana Press, New Jersey.