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| == Protocol for ''Streptomyces'' protein expression'''== | | == Protocol for ''Streptomyces'' protein expression'''== |
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| Plasmid pIJ6021 allows for high level protein production in ''Streptomyces'' species. This plasmid was first described by [https://www.sciencedirect.com/science/article/pii/0378111995005452?via%3Dihub Takano et al 1995] and utilises the pIJ101 origin of replication which leads to high copy number, sometimes approaching 100 copies per cell. It includes the thiostrepton inducible ''tipA'' promoter to induce expression of the target gene.
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| However, pIJ6021 lacks an ''E. coli'' replicon, meaning (i) any cloning must be done using ''Streptomyces'' spp.; and (ii) plasmid DNA cannot be transferred to ''Streptomyces'' spp. by conjugal transfer from ''E. coli''. These characteristics have likely limited the wide uptake of pIJ6021 for high level protein production, despite its proven utility and adoption by industry (unpublished).
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| The exclusion of an ''E. coli'' replicon in pIJ6021 was deliberate as observations by Bibb et al indicated an incompatibility when the two replicons are present on the same plasmid in ''Streptomyces'' spp. This leads to lower levels of transcription for derivatives containing an ''E. coli'' replicon and we have shown that this is due to the instability of the expression construct in ''Streptomyces'' species. This translates to lower protein production levels in ''Streptomyces'' species. Note however that plasmids containing both replicons are stable in ''E. coli''.
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| To circumvent this, we made derivative plasmids and developed a procedure that allows all cloning to be done in ''E. coli''. The origin of replication is then removed by restriction enzyme digestion and subsequent (re)circularization of rhe vector by ligation as a final step. The ligation mixture can then be used to transform ''Streptomyces'' protoplasts to generate strains for protein production. Thus far we have found ''Streptomyces lividans'' TK24 is the best host strain for protein production and we have used to produce several enzymes that are recalcitrant to production in ''E. coli''. Investigation of additional ''Streptomyces'' host strains is planned.
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| '''Notes:'''
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| When the ''E. coli'' replicon is removed from pIJ13110 by digestion with SpeI and (re)circularization it generates a version of pIJ6021 that differs by a very small number of nucleotide changes which do not change its biology.
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| The plasmid pIJ13111 is a modified version of pIJ13110 in which a C-terminal Hexa-histidine-tag has been incorporated into the resulting protein.
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| The work leading up to these plasmids will be published in due course, and further improvements to the system, including a version for N-terminal hexahistidine tagging are being generated and will be released when validated.
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| Add a Table with plasmid info for pIJ101, pIJ6021, pIJ13110 and pIJ 13111.
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| '''Cloning & protoplast transformation'''
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| 1. Digest the expression vector (pIJ13110 or pIJ13111) with NdeI and a second enzyme from HindIII, XbaI, BamHI or EcoRI in the MCS (we generally use NdeI-HindIII). DNA sequence maps of the plasmids are available on ActinoBase here.
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| We typically digest ~3 µg of DNA for 2-3 hours and dephosphorylate with claf alkaline phosphatase (CIP) to avoid (re)ligation of the vector.
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| 2. PCR amplify or synthesise the gene you wish to clone and then either:
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| Digest with the same enzymes you used to digest the vector. We use the same amount and time as with the vector. Purify the desired band from an agarose gel (we use the Qiaquick gel extraction kit from Qiagen). We normally use 75 ng of vector, and a vector to insert ratio of 1:3. We recommend using the 5U T4 DNA ligase for 1h+ because the sticky ends of NdeI are short.
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| Or use Gibson assembly, when generating your fragment by PCR include 20 nt overhangs (these have always been sufficient for us). Purify bands using an agarose gel. We normally use 60-70 ng of vector, and a vector to insert ratio of 1:2/1:3., and incubate for 1h at 50° C.
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| 3. Transform ''E. coli'' competent cells with the ligation or Gibson assembly mix. For ligations we use subcloning efficiency DH5 competent cells and for Gibson assemblies Electromax DH10B competent cells; both lines can be purchased. Select for transformants with apramycin (the kanamycin resistance gene in pIJ6021 does not work in ''E. coli''). Incubate the plates overnight at 37oC.
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| 4. Pick and check some transformant colonies. We designed two primers for E. coli colony PCR and sequencing.
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| • PtipA-fwd: GGATCTGGGCTGAGGGAG
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| • pIJ6021-check-rev: TTGTTGTCATTGTCGGCG
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| We generally use PtipA-fw plus the reverse primer of the gene in question for more specific amplification. For colony PCR we generally use Taq polymerase and an annealing temperature of 62° C. Check the construct is correct by purifying the plasmid and digesting with the restriction enzymes you used for cloning.
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| 5. '''KEY STEP!''' Prior to protoplast transformation remove the ''E. coli'' replicon by digesting approx. 3 µg of your construct with SpeI for 2-3h. Run the digest on a 1% agarose gel, cut out the top band (it contains everything but the ''E. coli'' replicon) and perform a standard ligation reaction to (re)circularise (Vf: 10 µl). This generates a plasmid backbone equivalent to plasmid pIJ60211.
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| 6. [[Protoplasts_Formation,_Regeneration_and_Transformation|Protoplast preparation and transformation]]
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| • To prepare protoplasts, we follow a modified version of this [[Protoplasts_Formation,_Regeneration_and_Transformation|protocol]]. Once prepared, distribute the protoplasts into 100 µl aliquots. They can be stored at -80° C for up to a year.
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| • For protoplast transformation, we use a modified version of the [[Protoplasts_Formation,_Regeneration_and_Transformation|PEG-assisted transformation procedure]]. Thaw an aliquot of ''S. lividans'' protoplasts on ice and mix with the 10 µl ligation ((re)circularization) reaction, then leave on ice for 2 min. Add 250 µl of PEG solution (1 g PEG6000 (we currently use material purchased from Fisher) in 3 ml P buffer (see manual page 415; sterile filtered) and leave on ice for 5 min. Plate the mixture on R5 agar plates (see manual page 409; 1 plate per transformation) and incubate at 30° C for 16-20h.
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| 7. Overlay with [[SNA]] (3 ml per plate) containing kanamycin (10 µg/ml) and thiostrepton (50 µg/ml); we use a pipette with 5 ml tip to achieve this evenly. Incubate at 30° C until colonies are of sufficient size to pick.
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| 8. Pick transformants and transfer onto [[SFM]] agar plates (page 409 manual; also known as MS agar) containing kanamycin (10 µg/ml). We also transfer the same colonies onto [[SFM]] agar plates containing apramycin (25 µg/ml) to check that the ''E. coli'' replicon has been removed correctly.
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| 9. Check strains by PCR using the PtipA-fwd and pIJ602-check-rev primers that anneal at each side of the cloning region.
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| '''Protein production culture conditions:'''
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| Initial test.
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| We tested multiple conditions, but the one that works best uses 100 ml Erlenmeyer flasks containing 34% [[YEME]] (25 ml) plus 2.5 g of 3 mm glass beads and kanamycin (final concentration = 10 µg/mL). Larger 250 ml Erlenmeyer flasks with springs also work but tend to give a slightly lower protein levels.
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| Inoculate each flask with spores scraped from a growing [[SFM]] agar plate using a toothpick and adding them to the medium. Incubate at 30° C and 200 rpm, and after 48h (OD600 0-4-0.8), induction is carried out by adding thiostrepton (to a final concentration of 20 µg/mL). Incubate for a further 48h under the same conditions.
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| To analyse protein levels, take 1 ml samples, spin them down, discard the supernatant and resuspend the pellets in 150 µl of lysis buffer containing 1 mg/ml lysozyme at 37° C for 2h. The lysis buffer is specific for each protein, and we typically start using 20 mM Tris and 100-500 mM NaCl. We sometimes find the addition of 0.4 M glucose was useful to help solubilise certain proteins. We check a range of pH’s between 7.2 and 9, starting with the calculated isoelectric point of the protein.
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| After the 2h incubation spin down the samples again to collect the supernatant (soluble protein) and resuspend the pellet in 350 µl of distilled H2O. Add blue SDS buffer (sample buffer) to all samples, boil for 15 min and run SDS-PAGE as you normally would.
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| '''Scaling up for protein purification.'''
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| For protein purification, we use 2L flasks containing springs and 500 ml of 34% [[YEME]] (foam can be controlled using antifoam once the incubation is finished); thiostrepton added as for the analytical scale experiments. The rest of the steps are the same, with the only difference being that after the 2h incubation with lysozyme, the mixture is sonicated for ca. 30 min (10s on, 10s off, amplitude 60%).
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| '''Appendix'''
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| To prepare protoplasts we grow a seed culture in [[TSB]] liquid media (50 ml Falcon tube with either small springs or few glass beads) and after 48 h transfer 250 µl of this to 250 ml Erlenmeyer flask containing springs and 25 ml 34% YEME (plus additives as per manual page 56). The rest of the procedure is as per the manual other than the aliquot volume, for which we use 100 µl as above.
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| Protoplast bath efficiency can be checked before transforming them if you desire. This involves transforming with an empty vector and plating a range of dilutions to quantify the number of transformants.
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