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Jul 2019
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High-level Production of Recombinant Membrane Proteins Using the Engineered Escherichia coli Strains SuptoxD and SuptoxR
利用工程大肠杆菌SuptoxD和SuptoxR高效生产重组膜蛋白   

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Abstract

We have previously described the development of two specialized Escherichia coli strains for high-level recombinant membrane protein (MP) production. These engineered strains, termed SuptoxD and SuptoxR, are capable of suppressing the cytotoxicity caused by MP overexpression and of producing greatly enhanced MP yields. Here, we present a Bio-protocol that describes gene overexpression and culturing conditions that maximize the accumulation of membrane-integrated and well-folded recombinant MPs in these strains.

Keywords: Recombinant protein production (重组蛋白生产), Membrane protein (膜蛋白), Toxicity (毒性), Escherichia coli (大肠杆菌), SuptoxD (SuptoxD), SuptoxR (SuptoxR)

Background

MPs perform a variety of critical functions in the cells of all living organisms (Wagner et al., 2006; Schlegel et al., 2010) and constitute major targets for current and future pharmaceuticals (Yildirim et al., 2007). Acquiring sufficient amounts of isolated protein is a prerequisite for their biochemical and structural studies, which in turn can lead to a deeper understanding of their functions and the discovery of new MP-targeting drugs.

Because MPs are typically encountered in their native environments at very low abundances, heterologous hosts have been routinely used for their recombinant overexpression and subsequent purification. Many different systems have been utilized as overexpression hosts for a large variety of MPs of both prokaryotic and eukaryotic origin (Wagner et al., 2006). Among these, Escherichia coli has been one of the most popular ones, owing to its very low cost and ease of use (Makino et al., 2011). Indeed, this bacterium has been successfully utilized for the production of approximately 20% of all recombinantly produced MP structures that are deposited in the Protein Data Bank (Dilworth et al., 2018). Despite these advantages and successes, the use of E. coli as a heterologous host for MP production is often accompanied by severe toxicity, low levels of final biomass and minute final yields (Miroux and Walker, 1996; Wagner et al., 2007; Link et al., 2008; Gubellini et al., 2011).

In order to address these challenges, we have recently developed two specialized E. coli strains, named SuptoxD and SuptoxR, which enable high-level production of recombinant MPs (Gialama et al., 2017a and 2017b). When used as expression hosts, these strains exert a dual positive effect compared to wild-type bacteria:
(1)They suppress the toxicity that frequently accompanies the MP overexpression process, thus enabling enhanced levels of final bacterial biomass, and
(2)They markedly increase the cellular accumulation of membrane-incorporated and properly folded protein.

Combined, these two positive effects result in dramatically enhanced volumetric yields for various recombinant MPs (Gialama et al., 2017a and 2017b). Importantly, these strains have been optimized to enable the production of high-quality recombinant MPs at quantities sufficient for functional and structural studies (Michou et al., 2019). Up to now, we have tested a broad panel of recombinant MPs of both prokaryotic and eukaryotic origin and with different characteristics, all of which are described in Gialama et al. (2017a and 2017b) and in Michou et al. (2019).

The toxicity-suppressing and cellular production-promoting capabilities of SuptoxD and SuptoxR are based on the overexpression of either one of the effector genes djlA or rraA, respectively (Gialama et al., 2017a). DjlA (DnaJ-like protein A) is a single-pass integral MP that functions mainly as a co-chaperone for the central bacterial molecular chaperone DnaK (Clarke et al., 1996). On the other hand, RraA (Regulator of ribonuclease activity A) is known to act as a regulator of the mRNA-degrading activity of RNase E, and rraA overexpression has been found to affect the levels of more than 2,000 different mRNAs in E. coli (Lee et al., 2003). We have previously found that (i) DjlA and RraA act independently, i.e., the beneficial effects of each protein on recombinant MP production occur through a mechanism that does not involve the other, and in a non-additive manner; (ii) full-length and membrane-bound DjlA is required for exerting its beneficial effects on recombinant MP production in E. coli SuptoxD; (iii) the MP production-promoting properties of DjlA in SuptoxD are mediated through the action of the molecular chaperone DnaK; (iv) the observed RraA-mediated effects in E. coli SuptoxR involve the ribonucleolytic activity of RNase E; and (v) DjlA and RraA are unique among similar E. coli proteins in their ability to promote bacterial recombinant MP production (Gialama et al., 2017b). The exact molecular mechanism with which DjlA and RraA suppress MP-induced toxicity and enhance recombinant MP production in E. coli SuptoxD and SuptoxR, respectively, is still under investigation.

Here, we present a protocol that describes gene overexpression and culturing conditions that maximize the accumulation of membrane-integrated and well-folded recombinant MPs when using SuptoxD and SuptoxR. As structural biology of MPs has entered a new era, we believe that these specialized strains will be broadly utilized to address some of the important challenges of MP production and will facilitate the acquirement of sufficient quantities of high-quality recombinant MPs.

Materials and Reagents

  1. Nylon-membrane Syringe filter 0.2 μm pore size, 25 mm diameter, sterile (Corning, catalog number: CLS431224 )
  2. HiLoad Superdex 200 16/600 column (GE Healthcare, catalog number: GE28-9893-35 )
  3. Sterile pipette tips, 10-200 µl (Greiner Bio, catalog number: 739290 )
  4. Sterile pipette tips, 200-1,000 µl (Greiner Bio, catalog number: 740290 )
  5. Sterile culture tubes (Sigma-Aldrich, catalog number: C1048-72EA )
  6. Sterile closures for culture tubes (Sigma-Aldrich, catalog number: C1298-100EA )
  7. Sterile centrifugation tubes 1.5 ml (Eppendorf, catalog number: 616201 )
  8. Sterile Falcon tubes 15 ml (Greiner Bio, catalog number: 188271 )
  9. Sterile Falcon tubes 50 ml (Greiner Bio, catalog number: 227261 )
  10. Semi-micro-cuvettes (Greiner, catalog number: 613101 )
  11. Polypropylene centrifuge bottles, 500 ml (Celltreat, catalog number: 229468 )
  12. Polycarbonate ultracentrifuge bottles, 3 ml (Beckman, catalog number: 355618 )
  13. Polypropylene chromatography columns, 5 ml (Pierce, catalog number: 29922 )
  14. Amicon® Ultra-15 Centrifugal Filter Unit (Merck, catalog number: UFC901024 )
  15. 96-well black plates for fluorescence measurements (Greiner, F-bottom, catalog number: 655076 )
  16. Polyvinylidene fluoride (PVDF) membrane (Merck, catalog number: IPVH00010 )
  17. E. coli SuptoxD cells
    Genotype: F- λΔ(ara-leu)7697 [araD139]B/r Δ(codB-lacI)3 galK16 galE15 e14- mcrA0 relA1 rpsL150(StrR) spoT1 mcrB1 hsdR2(r-m+) pSuptoxD
    Note: E. coli SuptoxD carry either the pSuptoxD or the pSuptoxD[untagged] plasmid (see below).
  18. E. coli SuptoxR cells
    Genotype: F- λ Δ(ara-leu)7697 [araD139]B/r Δ(codB-lacI)3 galK16 galE15 e14- mcrA0 relA1 rpsL150(StrR) spoT1 mcrB1 hsdR2(r-m+) pSuptoxR
    Note: E. coli SuptoxR carry either the pSuptoxR or the pSuptoxR[untagged] plasmid (see below).
  19. E. coli MC1061 cells (Coli Genetic Stock Center, catalog number: 6649 )
    Genotype: F- λ Δ(ara-leu)7697 [araD139]B/r Δ(codB-lacI)3 galK16 galE15 e14- mcrA0 relA1 rpsL150(StrR) spoT1 mcrB1 hsdR2(r-m+)
    Note: We have used E. coli MC1061 as a background host for the co-expression the djlA and rraA effector genes and for the evaluation of the performance of SuptoxD and SuptoxR extensively and successfully. However, we have found that the beneficial effects of DjlA and RrraA on MP productivity are independent of the use of MC1061 and that other E. coli K-12 and B strains can also be utilized as background hosts (Gialama et al., 2017a).
  20. Plasmid pASK-MP
    Notes:
    1. This plasmid can be generated by cloning a gene encoding a prokaryotic or eukaryotic MP into the pASK75 vector backbone (Biometra, Göttingen) (Skerra, 1994). We recommend flanking the MP’s sequence between the XbaI and anyone of the remaining restriction sites of the plasmid’s multiple cloning site (MCS), in order to remove the OmpA signal sequence.
    2. In order to facilitate MP purification via immobilized metal affinity chromatography (IMAC), we recommend inserting a poly-Histidine tag in-frame of the expressed MP. However, other tags and means of MP purification can be used according to the user’s particular preferences and needs.
    3. In order to easily monitor MP production, we recommend fusing the GFP reporter protein downstream of the MP of interest (Drew et al., 2001 and 2008).
    4. We have used the vector pASK-MP extensively for production of various recombinant MPs in E. coli SuptoxD and SuptoxR. However, we have found that the beneficial effects of these strains on MP productivity are independent of the use of the tet promoter of pASK75 (Gialama et al., 2017a). Thus, recombinant MPs can be overexpressed in these strains using other types of promoters and plasmids as well.
  21. pSuptoxD[untagged]
    Note: This plasmid overexpresses djlA, the gene encoding the E. coli membrane-bound DnaK co-chaperone DjlA, upon induction with L(+)-arabinose.
  22. pSuptoxD
    Notes:
    1. This plasmid overexpresses djlA, the gene encoding the E. coli membrane-bound DnaK co-chaperone DjlA, with a poly-histidine tag at its C-terminus, upon induction with L(+)-arabinose.
    2. Overexpression of the SuptoxD system can be monitored by western blotting using an anti-His antibody, according to standard protocols. The molecular weight of the expressed protein, i.e., DjlA-His6, is ~32 kDa.
    3. As we have found that the presence of the poly-histidine tag does not affect the activity of DjlA, we recommend using an untagged version of pSuptoxD, i.e., pSuptoxD[untagged], for MP purification via IMAC.
  23. pSuptoxR[untagged]
    Note: This plasmid overexpresses rraA, the gene encoding the inhibitor of the E. coli RNase E RraA, upon induction with L(+)-arabinose.
  24. pSuptoxR
    Notes:
    1. This plasmid overexpresses rraA, the gene encoding the inhibitor of the E. coli RNase E RraA, with a poly-histidine tag at its C-terminus upon induction with L(+)-arabinose.
    2. Overexpression of the SuptoxR system can be monitored by western blotting using an anti-His antibody, according to standard protocols. The molecular weight of the expressed protein, i.e., RraA-His6, is ~20 kDa.
    3. As we have found that the presence of the poly-histidine tag does not affect the activity of RraA, we recommend using an untagged version of pSuptoxR, i.e., pSuptoxR[untagged], for MP purification via IMAC.
  25. Sodium chloride for analysis, ACS, ISO (Applichem, catalog number: 131659 )
  26. Tryptone BioChemica BC (Applichem, catalog number: A1553 )
  27. Yeast extract BioChemica BC (Applichem, catalog number: A1552 )
  28. Agar bacteriology grade BC (Applichem, catalog number: A0949 )
  29. Ampicillin sodium salt (Sigma-Aldrich, catalog number: A9518 )
  30. Chloramplenicol (Sigma-Aldrich, catalog number: C0378 )
  31. L(+)-Arabinose (Applichem, catalog number: A9728 )
  32. Anhydrotetracycline hydrochloride (Sigma-Aldrich, catalog number: 37919 )
  33. Sodium hydroxide (NaOH)
  34. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
  35. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S7907 )
  36. Sodium dihydrogen phosphate, anhydrous (Chemlab, catalog number: CL00.1496 )
  37. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5655 )
  38. Tris Base (Fisher, catalog number: BP154-1 )
  39. Tween 20 (Fisher, catalog number: BP337 )
  40. Mini-PROTEAN TGX Precast 4-20% resolving gels (Bio-Rad, catalog number: 456-1094 )
  41. Non-fat dry milk (Sigma-Aldrich, catalog number: M7409 )
  42. Monoclonal anti-polyhistidine-peroxidase antibody produced in mouse (Sigma-Aldrich, catalog number: A7058 )
  43. Pierce ECL western blotting substrate kit (Thermo Fisher Scientific, catalog number: 32106 )
  44. n-dodecyl β-D-maltoside (DDM) (Glycon Biochemicals, catalog number: D97002 )
  45. Glycerol (Fisher, catalog number: G/0650/21 )
  46. Imidazole (Applichem, catalog number: A1073 )
  47. β-mercaptoethanol (Sigma, catalog number: M6250 )
  48. Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
  49. Ni-NTA Agarose (Qiagen, catalog number: 30230 )
  50. Ethanol absolute (ACROS Organics, catalog number: 448450025 )
  51. Luria-Bertani broth (LB) (see Recipes)
  52. Antibiotics (see Recipes)
  53. Inducers of protein production (see Recipes)
  54. Lysis buffer (see Recipes)
  55. Wash buffer (see Recipes)
  56. Elution buffer (see Recipes)
  57. Sodium dodecyl sulphate (SDS) sample buffer (6x) (see Recipes)
  58. Tris-buffered saline with Tween-20 (TBST) (see Recipes)
  59. Phosphate-buffered saline (PBS) (see Recipes)
  60. DDM solubilization buffer (see Recipes)
  61. Size exclusion buffer (see Recipes)

Equipment

  1. Centrifuge MiniSpin (Eppendorf, Mini Spin, catalog number: 5452000018 )
  2. High-speed refrigerated centrifuge (Kubota, catalog number: 7780 )
  3. Ultracentrifuge (Beckman Coulter, model: Optima LE-80K )
  4. Shaking incubator (Eppendorf, model: New BrunswickTM Innova® 44, catalog number: M1282-0006 )
  5. Roller mixer (Kisker, catalog number: L005-SLN )
  6. Microplate reader (Tecan, model: Safire II )
  7. Electrophoresis power supply (Consort, catalog number: EV231 )
  8. Imaging system for DNA and protein analysis (ChemiDoc-It2 Imaging System (UVP))
  9. Sonicator equipped with a 3 mm diameter probe (Qsonica, catalog number: Q125-110 )
  10. Spectrophotometer UV-VIS (Hitachi, model: U2000
  11. Mini PROTEAN Tetra Vertical Electrophoresis Cell (Bio-Rad, catalog number: 1658005 )
  12. Type 70Ti fixed-angle titanium rotor (Beckman, catalog number: 337922
  13. Homogenizer (Heidolph, model: RΖR1 )

Procedure

The use of the engineered E. coli strains SuptoxD and SuptoxR for the high-level production of recombinant MPs is outlined in Figure 1.


Figure 1. Overview of the E. coli SuptoxD and SuptoxR systems. The toxicity-suppressing and cellular production-promoting capabilities of E. coli SuptoxD and SuptoxR that result in dramatically enhanced volumetric yields for various recombinant MPs, are based on the overexpression of the E. coli genes djlA or rraA, respectively. The effector genes are overexpressed from the vectors pSuptoxD and pSuptoxR under the control of the araBAD promoter and its inducer L(+)-arabinose. For the production of recombinant MPs in these strains, we typically use pASK75-based plasmids under the control of a tet promoter and its inducer aTc (pASK-MP vector).

The procedure of the recombinant MP production and purification is outlined in Figure 2.


Figure 2. Overview of the recombinant MP production and purification procedure using the E. coli SuptoxD and SuptoxR strains. A. Transform either one of the SuptoxD or SuptoxR strains with the plasmid encoding the MP of interest. B. Overexpress the MP of interest together with the pSuptoxD or pSuptoxR system, at 25 °C for 16 h and using the appropriate inducers. C. Lyse cells by sonication. D. Centrifuge the total cell lysates and transfer the soluble supernatant into an ultracentrifugation tube. E. Ultracentrifuge the soluble cell lysate and collect the pelleted membranes. F. Mechanically resuspend the membranes in DDM-solubilization buffer using a homogenizer. G. Rotate for at least 1 h at 4 °C. H. Ultracentrifuge and collect the supernatant containing the solubilized membranes. I-J. Purify the solubilized membranes using IMAC and SEC.


  1. MP overexpression in shake flasks (Under sterile conditions)
    1. Transform (chemically or electro-) competent SuptoxD or SuptoxR cells with the pASK-MP vector and plate on LB agar containing 100 μg/ml ampicillin and 40 μg/ml chloramphenicol.
      Notes:
      1. When performing recombinant production of a specific MP of interest for the first time, we recommend using both the SuptoxD and SuptoxR strains, in order to determine the particular strain that yields higher levels of MP production.
      2. For the expression of highly toxic or otherwise difficult-to-express MPs that accumulate at very low yields, we highly recommend to use the pSuptoxD or pSuptoxR plasmid in isolated form and perform double transformation of pSuptoxD or pSuptoxR with the pASK-MP plasmid in fresh competent E. coli MC1061 cells. Other E. coli strains can also be used as hosts for the pSuptox vectors.
      3. We have used the pASK75 vector backbone for the expression of the MP of interest extensively. However, other expression vectors can also be used, provided that they are compatible with the pSuptoxD/R system (chloramphenicol resistance, p15A origin of replication, araBAD promoter) and preferably contain an inducible promoter with tight regulation.
    2. Pick a single transformed bacterial colony and inoculate liquid LB cultures supplemented with the selection antibiotics 100 μg/ml ampicillin and 40 μg/ml chloramphenicol for pASK-MP and SuptoxD/R system maintenance. Incubate at 37 °C for 16 h with constant shaking at 200-220 rpm.
      Notes:
      1. We highly recommend using freshly transformed E. coli cells for all MP production experiments in order to obtain maximum protein yields.
      2. The volume of the LB cultures should be enough for next-day’s inoculation.
    3. The following day prepare a 2% sub-culture in fresh LB supplemented with the appropriate antibiotics, as well as 0.01% or 0.2% (w/v) L(+)-arabinose for the induction of the SuptoxD and SuptoxR system respectively. Incubate at 30 °C with shaking until an optical density at 600 nm (OD600) of 0.5.
      Notes:
      1. The recommended L(+)-arabinose concentrations have been determined following expression optimization in SuptoxD/R using certain model recombinant MPs (Michou et al., 2019). Other MPs, however, may require other concentrations for their optimal accumulation.
      2. Keep working stock solution of arabinose (20% w/v) in aliquots to avoid contamination.
    4. Initiate overexpression of the target MP by adding 0.2 μg/ml anhydrotetracycline (aTc) and incubate overnight at 25 °C with shaking.
      Notes:
      1. We highly recommend using freshly prepared and light-protected aTc.
      2. We highly recommend initiating MP overexpression at an OD600 of precisely 0.5 as we have found that deviations result in lower protein yields.
      3. We recommend to not exceed 16 h of overexpression in order to minimize the accumulation of genomic/plasmid mutations that suppress MP production.
      4. The recommended aTc concentration has been determined following expression optimization in SuptoxD/R using certain model recombinant MPs (Michou et al., 2019). Other MPs, however, may require other concentrations for their optimal accumulation.
    5. Harvest cells by centrifugation at 4,000 x g for 10 min at 4 °C.
    6. Check for MP production by western blotting or activity assay according to standard protocols.
      Notes:
      1. Due to the MP-induced toxicity, it is not unusual for genomic/plasmid mutations to accumulate and bypass the overproduction of MP. This will result in a high increase of the culture’s OD600 and low yield of MP production. For this reason, MP production should always be checked before continuing with MP purification.
      2. When overexpressing an MP-GFP fusion, the bacterial fluorescence can be used as an indicator of protein production. In this case, cells corresponding to an OD600 = 1 should be resuspended in 100 μl PBS and fluorescence should be measured:
        1. Using a 96-well plate reader at 510 nm after excitation at 488.
        2. Using a flow cytometer equipped with a 488 nm solid state laser for excitation and a 530/30 nm band pass filter for detection. In this case, events should be first gated in a forward scatter (FSC) vs. side scatter (SSC) plot in order to eliminate non-cellular events and then plotted in a histogram of fluorescence intensity detected by the FITC (fluorescein isothiocyanate) channel photomultiplier tube (PMT) vs. number of events, in order to estimate the mean fluorescence of the population.

  2. Bacterial membrane isolation and MP extraction
    1. Resuspend a cell pellet from a 1 L culture in 10 ml ice-cold lysis buffer.
    2. Lyse cells by sonication for 4 x 25 s on ice, with a 1 min interval between each sonication.
    3. Centrifuge the total cell lysates (10,000 x g, 15 min, 4 °C) and transfer carefully the soluble supernatant into an ultracentrifugation tube.
    4. Ultracentrifuge the soluble fraction at 130,000 x g for 1 h at 4 °C using a 70-Ti rotor, in order to pellet the bacterial membranes.
    5. Collect the pelleted membranes using a spatula and mechanically resuspend them in 5 ml ice-cold DDM-solubilisation buffer using a homogenizer. The homogenate should contain no visible particles.
      Note: Care should be taken when collecting the membranes as they form a very sticky and difficult to handle pellet.
    6. Rotate the resuspension at 180 rpm for at least 1 h at 4 °C, in order for MP extraction to take place.
      Note: A range of different DDM concentrations and incubation periods should be tested in order to determine the optimal conditions for MP extraction. When using MP-GFP fusions or chromophore-containing MPs, such as certain bacteriorhodopsins (Michou et al., 2019), this procedure can be easily optimized by regular centrifugations and monitoring of the fluorescence levels or discoloration respectively, of the resulting pellet.
    7. Ultracentrifuge at 130,000 x g for 1 h at 4 °C and collect the supernatant that contains the extracted MP.
    8. Check for successful MP extraction by western blotting or activity assay according to standard protocols.
      Note: When overexpressing an MP-GFP fusion, we highly recommend using in-gel fluorescence analysis as a quality control of the extraction of well-folded and active MP (Geertsma et al., 2008). In this case, the following procedure should be followed:
      1. Mix 100 μl of the supernatant containing the extracted MP with 20 μl of 6x SDS loading dye.
      2. Load 50 μl of sample on a 4-20% precast polyacrylamide gel without prior boiling of the sample and run the gel at a constant voltage of 80 V.
      3. Visualize the MP-GFP fluorescent band on an imaging system, e.g., ChemiDoc-It2 Imaging System, equipped with a CCD camera and a GFP filter, after exposure of about 3 s.
      4. Western blotting of the analysed proteins according to standard protocols should result in the visualization of the MP’s dual band migration, with the lower band corresponding to the fluorescent and well-folded MP-GFP fusion and the upper band corresponding to non-fluorescent and misfolded MP-GFP fusion (Drew et al., 2008; Geertsma et al., 2008).
      5. Analyzing the same samples after boiling for 10 min with SDS-PAGE and western blotting results in full denaturation of the fusion and single band migration that lacks fluorescence.
    9. Store the supernatant on ice until further use or purification.

  3. Small-scale, two-step MP purification for initial studies using immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC) (All steps should be performed at 4 °C)
    1. Transfer 1 ml of Ni-NTA agarose resin to a 15 ml tube and remove the supernatant after brief centrifugation at low speed (1,000 x g, 1 min). Resuspend in 2 ml lysis buffer and mix by gently inverting.
    2. Mix the supernatant containing the extracted MP with the equilibrated resin from the above step at 4 °C for 1 h on a roller mixer.
    3. Equilibrate a 5 ml polypropylene chromatography column with 1 column volume (CV) of lysis buffer and load the mixture onto the equilibrated column.
    4. Collect the flow-through, and reload it onto the column. Repeat for a total of 5-6 times.
      Note: This step should be repeated multiple times in order to ensure complete binding of the MP to the nickel resin. When using MP-GFP fusions or chromophore-containing MPs, this step should be repeated until a non-fluorescent or colourless flow-through is collected.
    5. Wash the column-bound protein with 40 ml wash buffer.
    6. Elute the MP with 6 ml elution buffer.
    7. Concentrate the elution to a final volume of approximately 500 μl using a centrifugal filter.
    8. Equilibrate a Superdex 200 column with 2 CV of size exclusion buffer at 1 ml/min, ensuring that the pressure does not exceed the maximum limit of 0.3 MPa.
    9. Inject the concentrated sample onto the column and pass 1 CV of size exclusion buffer through the system while collecting the eluted fractions. 
    10. Pool together the fractions that correspond to the MP’s peak.
    11. Quantify the amount of purified MP by calculating the absorbance at 280 nm of the integrated peak area and using the MP’s extinction coefficient (ε280).
      Note: By following this protocol, and using the SuptoxD strain we have obtained 1 mg per L of bacterial culture of purified human bradykinin receptor 2 (BR2), which under normal conditions exhibits very high levels of toxicity and low yields when overexpressed in E. coli (Michou et al., 2019). This amount was 14-fold higher than the corresponding yield from wild-type E. coli. Similar results were achieved by the use of SuptoxR in overexpressing the large bacterial mechano-sensitive ion channel (MscL), in which case we isolated 0.33 mg per L of bacterial shake flask culture, a > 10-fold increased yield compared to wild-type E. coli (Michou et al., 2019). However, other means of MP purification can be used according to the user’s particular preferences and needs.
    12. Evaluate MP purity by SDS-PAGE analysis and Coomassie staining.
      Note: For highly concentrated MP fractions, we recommend running the gel at a very low constant current, such as 12-13 mA.

Recipes

  1. Luria-Bertani broth (LB)
    10 g/L tryptone
    5 g/L yeast extract
    10 g/L NaCl
    Adjust pH to 7.0 with 5 N NaOH
    Autoclave for sterilization for 20 min at 15 psi
  2. Antibiotics
    1. 40 mg/ml chloramphenicol in ethanol 99.8%, store at -20 °C
    2. 100 mg/ml ampicillin in MilliQ water, sterilize the solution using a 0.22 μm syringe filter, store at -20 °C
  3. Inducers of protein production
    1. 20% w/v L(+)-arabinose in MilliQ water, sterilize the solution using a 0.22 μm syringe filter, store at room temperature
    2. 20 mg/L anhydrotetracycline (aTc) in ethanol 99.8%, store at -20 °C
  4. Lysis buffer
    300 mM NaCl
    50 mM NaH2PO4
    15% glycerol
    5 mM dithiothreitol (add immediately before use)
    0.1 mM PMSF (add immediately before use)
    Adjust pH to 7.5
    Store at 4 °C
  5. Wash buffer
    20 mM imidazole
    300 mM NaCl
    50 mM NaH2PO4
    Adjust pH to 8
    Add 0.1% w/v DDM (add immediately before use)
    Store at 4 °C
  6. Elution buffer
    250 mM imidazole
    300 mM NaCl
    50 mM NaH2PO4
    Adjust pH to 8
    Add 0.1% w/v DDM (add immediately before use)
    Store at 4 °C
  7. Sodium dodecyl sulphate (SDS) sample buffer (6x)
    6% w/v SDS
    300 mM Tris
    15% v/v glycerol
    0.01% w/v bromophenol blue
    10% v/v β-mercaptoethanol
    Adjust pH to 6.8
  8. Tris-buffered saline with Tween-20 (TBST)
    20 mM Tris base
    150 mM NaCl
    Adjust pH to 7.5
    0.1% v/v Tween-20
  9. Phosphate-buffered saline (PBS)
    137 mM NaCl
    2.7 mM KCl
    10 mM Na2HPO4
    1.8 mM KH2PO4
    Adjust pH to 7.4
    Autoclave for sterilization for 20 min at 15 psi
  10. DDM solubilization buffer
    Lysis buffer
    2.5% w/v DDM (add immediately before use)
  11. Size exclusion buffer
    This buffer should be selected according to the characteristics of the MP of interest. Some examples include:
    PBS supplemented with 0.1% w/v DDM or 10 mM HEPES, 0.4 M NaCl, 0.05% w/v DDM, pH 7.2

Acknowledgments

This work has received funding from the following: (i) the Greek State Scholarships Foundation (Idryma Kratikon Ypotrofion–IKY) scholarship, funded by the action “Strengthening human research potential through doctoral research” of the Partnership Agreement “Development of human potential, education and lifelong learning” 2014-2020 (Grant number: MIS 5000432), which is co-financed by the European Structural and Investment Fund (ESIF) and the Greek State, (ii) the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (Project “ProMiDis”; grant agreement no. 819934).

Competing interests

GS is inventor on a patent application for E. coli SuptoxD and SuptoxR (PCT/EP2017/025168). The authors declare no other competing interests.

References

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  2. Dilworth, M. V., Piel, M. S., Bettaney, K. E., Ma, P., Luo, J., Sharples, D., Poyner, D. R., Gross, S. R., Moncoq, K. and Henderson, P. J. (2018). Microbial expression systems for membrane proteins. Methods 147: 3-39. 
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简介

[摘要] 我们之前已经描述了两种用于生产高水平重组膜蛋白(MP)的大肠杆菌菌株的开发。这些工程菌株,称为SuptoxD和SuptoxR,能够抑制MP过度表达引起的细胞毒性,并产生显著提高的MP产量。在这里,我们提出一个生物协议,描述基因过度表达和培养条件,最大限度地积累膜整合和折叠良好的重组多磺酸粘多糖在这些菌株。

[背景]多磺酸粘多糖在所有活生物体的细胞中执行多种关键功能(Wagner et al.,2006;Schlegel et al.,2010),是当前和未来药物的主要靶点(Yildrim et al.,2007)。获得足够数量的分离蛋白是进行生化和结构研究的前提,这反过来又可以加深对其功能的理解,并发现新的MP靶向药物。

由于多磺酸粘多糖通常在其天然环境中以极低的丰度出现,异源宿主通常用于其重组过表达和随后的纯化。许多不同的系统已被用作原核和真核来源的多种多磺酸粘多糖的过表达宿主(Wagner等人,2006年)。其中,大肠杆菌是最受欢迎的一种,因为它的成本非常低,使用方便(Makino等人,2011年)。事实上,这种细菌已经成功地用于生产储存在蛋白质数据库中的所有重组产生的MP结构的大约20%(Dilworth等人,2018年)。尽管有这些优势和成功,但使用大肠杆菌作为MP生产的异源宿主通常伴随着严重的毒性、低水平的最终生物量和微小的最终产量(Miroux和Walker,1996;Wagner等人,2007;Link等人,2008;Gubellini等人,2011)。

为了应对这些挑战,我们最近开发了两种专门的大肠杆菌菌株,分别命名为SuptoxD和SuptoxR,它们能够高水平地生产重组多磺酸粘多糖(Gialama等人,2017a和2017b)。当用作表达宿主时,与野生型细菌相比,这些菌株具有双重积极作用:

(1) 因此,MP的过度表达常常会增强细菌的毒性

(2) 它们能显著增加细胞膜结合和适当折叠的蛋白质的积累。

这两种积极作用结合在一起,可显著提高各种重组多磺酸粘多糖的体积产量(Gialama等人,2017a和2017b)。重要的是,这些菌株已经过优化,能够以足够的数量生产高质量的重组多磺酸粘多糖,用于功能和结构研究(Michou等人,2019年)。到目前为止,我们已经测试了大量的原核和真核来源的具有不同特性的重组多磺酸粘多糖,所有这些都在贾拉马等人的研究中进行了描述。(2017a和2017b)以及Michou等人。(2019年)。

SuptoxD和SuptoxR的毒性抑制和细胞生产促进能力分别基于效应基因djlA或rraA的过度表达(Gialama等人,2017a)。DjlA(DnaJ-like protein A)是一种单程完整的MP,主要作为中心细菌分子伴侣DnaK的辅伴侣(Clarke al.,1996)。另一方面,RraA(核糖核酸酶活性调节器A)被认为是RNase E mRNA降解活性的调节器,RraA过表达可影响大肠杆菌中超过2000种不同的mRNA水平(Lee等人,2003年)。我们以前发现(i)DjlA和RraA是独立作用的,即每种蛋白对重组MP产生的有益作用是通过一种不涉及另一种的机制发生的,并且是以非加性的方式发生的;(ii)在大肠杆菌SuptoxD中发挥其对重组MP生产的有益作用需要全长和膜结合的DjlA;(iii)SuptoxD中DjlA促进MP产生的特性是通过分子伴侣DnaK的作用来实现的;(iv)在大肠杆菌SuptoxR中观察到的RraA介导的作用涉及RNase E的核糖核溶解活性;并且(v)DjlA和RraA在促进细菌重组MP产生的能力方面是独一无二的(Gialama等人,2017b)。DjlA和RraA分别在大肠杆菌SuptoxD和SuptoxR中抑制MP诱导的毒性和提高重组MP产量的确切分子机制仍在研究中。et公司

在这里,我们提出了一个方案,它描述了基因过度表达和培养条件,当使用SuptoxD和SuptoxR时,最大限度地累积膜整合和折叠良好的重组多磺酸粘多糖。随着多磺酸粘多糖的结构生物学已经进入了一个新的时代,我们相信这些专用菌株将被广泛应用于解决多磺酸粘多糖生产的一些重要挑战,并将有助于获得足够数量的高质量重组多磺酸粘多糖。

关键字:重组蛋白生产, 膜蛋白, 毒性, 大肠杆菌, SuptoxD, SuptoxR

材料和试剂


 


1尼龙膜注射器过滤器0.21尼龙膜注射器过滤器0.2μm孔径,25 mm直径,无菌(康宁,目录号:CLS431224)


2.     HiLoad Superdex 200 16/600列(GE Healthcare,目录号:GE28-9893-35)


3.     无菌移液管头,10-200µl(Greiner Bio,目录号:739290)


4.     无菌吸管头,200-1000µl(Greiner Bio,目录号:740290)


5.     无菌培养管(Sigma-Aldrich,目录号:C1048-72EA)


6.     培养管无菌瓶塞(Sigma-Aldrich,目录号:C1298-100EA)


7.     无菌离心管1.5 ml(Eppendorf,目录号:616201)


8.     无菌猎鹰试管15毫升(Greiner Bio,目录号:188271)


9.     无菌Falcon试管50毫升(Greiner Bio,目录号:227261)


10.  半微量反应杯(Greiner,目录号:613101)


11.  聚丙烯离心瓶,500毫升(Celltreat,目录号:229468)


12.  聚碳酸酯超离心瓶,3毫升(贝克曼,目录号:355618)


13.  聚丙烯色谱柱,5ml(皮尔斯,目录号:29922)


14.  Amicon®Ultra-15离心过滤装置(默克公司,产品目录号:UFC901024)


15.  96孔黑色荧光测量板(Greiner,F-bottom,目录号:655076)


16.  聚偏氟乙烯(PVDF)膜(默克公司,产品目录号:IPVH00010)


17.  大肠杆菌SuptoxD细胞


基因型:F-λ-Δ(ara leu)7697[araD139]B/rΔ(codB-lacI)3 galK16 galE15 e14-mcrA0 relA1 rpsL150(StrR)spoT1 mcrB1 hsdR2(r-m+)pSuptoxD


注:大肠杆菌SuptoxD携带pSuptoxD或pSuptoxD[未标记]质粒(见下文)。


18.  大肠杆菌SuptoxR细胞


基因型:F-λ-Δ(ara leu)7697[araD139]B/rΔ(codB-lacI)3 galK16 galE15 e14-mcrA0 relA1 rpsL150(StrR)spoT1 mcrB1 hsdR2(r-m+)pSuptoxR


注:大肠杆菌SuptoxR携带pSuptoxR或pSuptoxR[untagged]质粒(见下文)。


19.  大肠杆菌MC1061细胞(大肠杆菌基因储备中心,目录号:6649)


基因型:F-λ-Δ(ara leu)7697[araD139]B/rΔ(codB-lacI)3 galK16 galE15 e14-mcrA0 relA1 rpsL150(StrR)spoT1 mcrB1 hsdR2(r-m+)


注:我们已将大肠杆菌MC1061作为背景宿主,用于共表达djlA和rraA效应基因,并广泛而成功地评价了SuptoxD和SuptoxR的性能。然而,我们发现,DjlA和RrraA对MP生产力的有益影响独立于MC1061的使用,其他大肠杆菌K-12和B菌株也可作为背景宿主使用(Gialama等人,2017a)。


20.  质粒pASK-MP


笔记:


a。这种质粒可以通过将编码原核或真核MP的基因克隆到pASK75载体主干(Biometra,Göttingen)来生成(Skerra,1994)。我们建议在XbaI和质粒多克隆位点(MCS)的任何一个剩余限制位点之间侧翼MP序列,以去除OmpA信号序列。


b。为了便于通过固定化金属亲和层析(IMAC)纯化MP,我们建议在表达的MP框架中插入一个聚组氨酸标签。然而,其他的标签和MP净化的方法可以根据用户的特殊偏好和需求使用。


c。为了便于监测MP的产生,我们建议融合相关MP下游的GFP报告蛋白(Drew等人,2001年和2008年)。


d。我们已经广泛地使用载体pASK-MP在大肠杆菌SuptoxD和SuptoxR中生产各种重组MPs。然而,我们发现这些菌株对MP生产力的有益影响与使用pASK75的tet启动子无关(Gialama等人,2017a)。因此,重组多磺酸粘多糖可以在这些菌株中使用其他类型的启动子和质粒过度表达。


21.  pSuptoxD[未标记]


注:这个质粒结束了在L(+)-阿拉伯糖诱导下,表达编码大肠杆菌膜结合DnaK辅伴侣djlA的基因djlA。


22.  pSuptoxD公司


笔记:


a。这个质粒结束了在L(+)-阿拉伯糖诱导下,表达编码大肠杆菌膜结合DnaK辅伴侣djlA的基因djlA,其C端带有聚组氨酸标签.


b。过度表达根据标准方案,SuptoxD系统可通过免疫印迹法使用抗His抗体进行监测。表达的蛋白质,即DjlA-His6的分子量为~32kda。


c。由于我们发现聚组氨酸标签的存在不会影响DjlA的活性,我们建议使用未标记的pSuptoxD版本,即pSuptoxD[未标记],用于通过IMAC纯化MP。


23.  pSuptoxR[未标记]


注:这个质粒结束了在L(+)-阿拉伯糖诱导下表达rraA,编码大肠杆菌RNase E rraA抑制剂的基因.


24.  pSuptoxR公司


笔记:


a。这个质粒结束了在L(+)-阿拉伯糖诱导下,表达编码大肠杆菌核糖核酸酶E rraA抑制剂的基因rraA,其C端带有一个聚组氨酸标签.


b。过度表达根据标准方案,SuptoxR系统可通过免疫印迹法使用抗His抗体进行监测。表达的蛋白质,即RraA-His6的分子量约为20kda。


c。由于我们发现聚组氨酸标签的存在不会影响RraA的活性,我们建议使用未标记的pSuptoxR,即pSuptoxR[未标记],通过IMAC进行MP纯化。


25.  分析用氯化钠,ACS,ISO(Applichem,目录号:131659)


26胰蛋白胨生化26胰蛋白胨生化BC(Applichem,目录号:A1553)


27酵母抽提物生化27酵母抽提物生化BC(Applichem,目录号:A1552)


28.  BC级琼脂细菌学(Applichem,目录号:A0949)


29.  氨苄西林钠盐(Sigma-Aldrich,目录号:A9518)


30.  氯霉素(Sigma-Aldrich,目录号:C0378)


31.  L(+)-阿拉伯糖(Applichem,目录号:A9728)


32.  盐酸脱水四环素(Sigma-Aldrich,目录号:37919)


33.  氢氧化钠(NaOH)


34.  氯化钾(KCl)(Sigma-Aldrich,目录号:P9333)


35.  磷酸二钠(Na2HPO4)(Sigma-Aldrich,目录号:S7907)


36.  无水磷酸二氢钠(Chemlab,目录号:CL00.1496)


37.  磷酸二氢钾(KH2PO4)(Sigma-Aldrich,目录号:P5655)


38.  Tris Base(Fisher,目录号:BP154-1)


39.  吐温20(费希尔,目录号:BP337)


40.  Mini-PROTEAN TGX预制4-20%分辨率凝胶(Bio-Rad,目录号:456-1094)


41.  脱脂奶粉(Sigma-Aldrich,目录号:M7409)


42.  小鼠抗多组氨酸过氧化物酶单克隆抗体(Sigma-Aldrich,目录号:A7058)


43.  皮尔斯ECL western-blotting基板试剂盒(赛默飞世尔科技公司,目录号:32106)


44.  正十二烷基β-D-麦芽糖苷(DDM)(甘醇生化制品,目录号:D97002)


45.  甘油(Fisher,目录号:G/0650/21)


46.  咪唑(Applichem,目录号:A1073)


47.  β-巯基乙醇(Sigma,目录号:M6250)


48.  苯基甲基磺酰氟(PMSF)(Sigma-Aldrich,目录编号:P7626)


49.  Ni-NTA琼脂糖(Qiagen,目录号:30230)


50.  无水乙醇(ACROS Organics,目录号:448450025)


51.  鲁里亚贝塔尼肉汤(LB)(见食谱)


52.  抗生素(见处方)


53.  蛋白质生产诱导剂(见配方)


54.  溶解缓冲液(见配方)


55.  洗涤缓冲液(见配方)


56.  洗脱缓冲液(见配方)


57.  十二烷基硫酸钠(SDS)样品缓冲液(6x)(见配方)


58.  Tris缓冲盐水与吐温-20(TBST)(见配方)


59.  磷酸盐缓冲盐水(PBS)(见配方)


60.  DDM增溶缓冲液(见配方)


61.  大小排除缓冲区(见配方)


 


设备


 


1.     微型离心机(Eppendorf,Mini-Spin,目录号:545200018)


2.     高速冷冻离心机(Kubota,目录号:7780)


3.     超离心机(Beckman Coulter,型号:Optima LE-80K)


4.     摇动培养箱(Eppendorf,型号:New BrunswickTM Innova®44,目录号:M1282-0006)


5.     辊式搅拌机(Kisker,目录号:L005-SLN)


6.     微板阅读器(帝肯,型号:Safire II)


7.     电泳电源(Consort,目录号:EV231)


8.     DNA和蛋白质分析成像系统(ChemiDoc-It2成像系统(UVP))


9.     配备3 mm直径探头的声波仪(Qsonica,目录号:Q125-110)


10.  紫外可见分光光度计(日立,型号:U2000)


11.  微型蛋白四垂直电泳池(Bio-Rad,目录号:1658005)


12.  70Ti型固定角钛转子(Beckman,目录号:337922)


13.  Olr1,均质机型号:


 


程序


 


工程大肠杆菌SuptoxD和SuptoxR用于高水平生产重组多磺酸粘多糖的用途如图1所示。






图1。大肠杆菌SuptoxD和SuptoxR系统概述。大肠杆菌SuptoxD和SuptoxR的毒性抑制和促进细胞生产的能力使各种重组多磺酸粘多糖的体积产量显著提高,这分别基于大肠杆菌基因djlA或rraA的过度表达。在araBAD启动子及其诱导物L(+)-阿拉伯糖的控制下,载体pSuptoxD和pSuptoxR的效应基因过度表达。为了在这些菌株中生产重组多磺酸粘多糖,我们通常在tet启动子及其诱导子aTc(pASK-MP载体)的控制下使用基于pASK75的质粒。




 


重组MP的生产和纯化程序如图2所示。


 






图2。利用大肠杆菌SuptoxD和SuptoxR菌株生产和纯化重组MP的方法综述。A、 用编码MP的质粒转化SuptoxD或SuptoxR菌株。B、 在25°C下,使用适当的诱导剂,将感兴趣的MP与pSuptoxD或pSuptoxR系统一起过度表达16小时。C、 用超声波溶解细胞。D、 离心总细胞裂解物并将可溶性上清液转移到超速离心管中。E、 用超速离心法将可溶性细胞溶解并收集成膜。F、 使用均质器在DDM溶解缓冲液中机械地再悬浮膜。G、 在4℃下旋转至少1h。超速离心并收集含有溶解膜的上清液。I-J.用IMAC和SEC纯化增溶膜。


 


A、 MP在摇瓶中的过度表达(无菌条件下)


1.     用pASK-MP载体和平板在含有100μg/ml氨苄西林和40μg/ml氯霉素的LB琼脂上转化(化学或电)有能力的SuptoxD或SuptoxR细胞。


笔记:


a。当第一次进行特定MP的重组生产时,我们建议同时使用SuptoxD和SuptoxR菌株,以确定产生更高水平MP的特定菌株。


b。对于以极低的产量积累的剧毒或其他难以表达的MPs的表达,我们强烈建议以分离形式使用pSuptoxD或pSuptoxR质粒,并使用帕斯克MP质粒在新鲜大肠杆菌MC1061细胞中的表达。其他大肠杆菌也可作为pSuptox载体的宿主。


c。我们广泛使用了pASK75载体主干来表达感兴趣的MP。然而,也可以使用其他表达载体,只要它们与pSuptoxD/R系统兼容(氯霉素抗性,p15A复制起源,araBAD启动子),并且最好包含具有严格调控的n诱导启动子。


2.     选择一个转化菌群,接种液体LB培养液,添加100μg/ml氨苄西林和40μg/ml氯霉素,以维持pASK-MP和SuptoxD/R系统。以220-200转/分恒速振荡培养16小时。摄氏度


笔记:


a。我们强烈建议在所有MP生产实验中使用新转化的大肠杆菌细胞,以获得最大的蛋白质产量。


b。LB培养物的体积应足够第二天接种。


3.     第二天在新鲜LB中准备2%的亚培养基,并添加适当的抗生素,以及0.01%或0.2%(w/v)L(+)-阿拉伯糖,分别用于诱导SuptoxD和SuptoxR系统。在30°C下摇动培养,直到600 nm(OD600)的光密度为0.5。


笔记:


a。已使用以下推荐的Michou/supl模型测定了重组MPs(+)的浓度。然而,其他多磺酸粘多糖可能需要其他浓度才能达到最佳积累。


b。阿拉伯糖(20%)储备液 以避免污染。


4.     通过添加0.2μg/ml酸酐四环素(aTc)来启动靶点MP的过度表达,并在25℃下摇床培养过夜。


笔记:


a。我们强烈建议使用新制备的光保护aTc。


b。我们强烈建议在OD600精确到0.5开始MP过度表达,因为我们发现偏差会导致蛋白质产量降低。


c。我们建议尽量减少MP/h基因突变的产生。


d。T他推荐的aTc浓度是在使用某些模型重组MPs在SuptoxD/R中优化表达后确定的(Michou等人,2019年)。然而,其他多磺酸粘多糖可能需要其他浓度才能达到最佳积累。


5.     通过在4000 x g下在4°C下离心10分钟来收获细胞。


6.     根据标准方案,用免疫印迹法或活性分析法检查是否有MP产生。


笔记:


a。由于MP引起的毒性,因此基因组/质粒突变以积累和绕过MP的过量生产。ODMP高产栽培可提高产量。因此,在继续进行MP净化之前,应始终检查MP的生产情况。


b。当MP-GFP融合蛋白过度表达时,细菌荧光可作为蛋白质产生的指标。在这种情况下,对应于OD600=1的单元应在100内重新悬浮微升应测量PBS和荧光:


一。在488激发后,在510 nm处使用96孔板阅读器。


二。使用配备的流式细胞仪具有488nm固体激光器激发,530/30nm带通滤波器检测。在这种情况下,事件应首先在前向散射(FSC)与侧面散射(SSC)图中选通以消除非细胞事件,然后绘制FITC(异硫氰酸荧光素)通道光电倍增管(PMT)检测到的荧光强度与事件数的直方图中,为了估计群体的平均荧光。


 


B、 细菌膜分离与MP提取


1.     将1L培养物中的细胞颗粒重新悬浮在10 ml冰冷裂解缓冲液中。


2.     在冰上用超声波溶解细胞4 x 25 s,每次超声间隔1分钟。


3.     离心总细胞裂解物(10000 x g,15 min,4°C),并小心地将可溶上清液转移到超速离心管中。


4.     使用70 Ti转子在4°C下以130000 x g的速度将可溶部分超速离心1小时,以使细菌膜颗粒化。


5.     使用抹刀收集颗粒膜,并使用均质器机械地将其重新悬浮在5 ml冰镇DDM溶解缓冲液中。匀浆中不应含有可见颗粒。


注意:收集膜时应小心,因为它们会形成一个非常粘且难以处理的颗粒。


6.     在4°C下以180 rpm转速旋转再悬浮至少1小时,以便进行MP提取。


注:为了确定MP提取的最佳条件,应测试一系列不同的DDM浓度和孵育期。当使用某些含荧光素的荧光素或紫红质时s(Michou等人,2019年),这一程序可以很容易地优化通过定期离心和监测荧光水平或脱色,得到的小球。


7.     以130000 x g在4°C下超离心1h,并收集含有提取MP的上清液。


8.     根据标准方案,通过western印迹法或活性分析法检查是否成功提取MP。


注:当过度表达MP-GFP融合时,我们强烈建议使用凝胶内荧光分析作为提取折叠良好且活性MP的质量控制(Geertsma等人,2008年). 在这种情况下,应遵循以下程序:


a、 将100μl含有提取MP的上清液与20μl 6x SDS负载染料混合。


b、 在4-20%的预制聚丙烯酰胺凝胶上加载50μl样品,无需事先煮沸样品,并在80 V的恒定电压下运行凝胶。


c、 在曝光约3秒后,在成像系统上显示MP-GFP荧光带,例如ChemiDoc-It2成像系统,配备CCD摄像头和GFP过滤器。


d、 根据标准方案对分析的蛋白质进行Western blotting分析后,应能观察到MP的双带迁移,较低的带对应于荧光和折叠良好的MP-GFP融合,而上带对应于非荧光和错误折叠的MP-GFP融合(Drew et al.,2008;Geertsma et al。,2008年)。


e、 同一样品煮沸10min后用SDS-PAGE和western印迹分析,融合完全变性,单带迁移,缺乏荧光。


9.     将上清液储存在冰上,直到进一步使用或净化。


 


C、 使用固定化金属亲和层析(IMAC)和大小排阻色谱法(SEC)进行初步研究的小规模两步MP纯化(所有步骤均应在4℃下进行)


1.     将1ml Ni-NTA琼脂糖树脂转移至15ml试管中,在低速(1000xg,1min)短暂离心后去除上清液。在2ml溶解缓冲液中再悬浮,轻轻翻转混合。


2.     将含有提取的MP的上清液与上述步骤中的平衡树脂在4°C下在滚筒式混合器上混合1h。


3.     使5ml聚丙烯色谱柱与1柱体积(CV)的裂解缓冲液平衡,并将混合物加载到平衡柱上。


4.     收集流经的液体,并将其重新加载到柱上。重复5-6次。


注:该步骤应重复多次,以确保MP与镍树脂完全结合。当使用MP-GFP熔丝或含有MPs的生色团时,应重复此步骤,直到收集到非荧光或无色的流动。


5.     用40ml洗涤缓冲液洗涤柱结合蛋白。


6.     用6毫升洗脱缓冲液洗脱MP。


7.     使用离心过滤器将洗脱液浓缩至约500μl的最终体积。


8.     以1ml/min的速度使Superdex 200色谱柱与2 CV大小的排阻缓冲液平衡,确保压力不超过0.3MPa的最大限值。


9.     将浓缩样品注入柱上,在收集洗脱部分的同时,将1cv大小的排除缓冲液通过系统。


10.  把对应于议员峰值的分数集中起来。


11.  通过计算积分峰面积280 nm处的吸光度并使用MP的消光系数(ε280)来量化纯化MP的量。


注:通过遵循此方案,并使用SuptoxD菌株,我们获得了每升1毫克的纯化人缓激肽受体2(BR2)的细菌培养物,在正常条件下,当在大肠杆菌中过度表达时,该受体表现出非常高的毒性和较低的产量(Michou等人,2019年)。这一数量是野生型大肠杆菌相应产量的14倍。使用SuptoxR过度表达大细菌机械敏感离子通道(MscL)也获得了类似的结果,在这种情况下,我们分离出0.33 mg/L的细菌摇瓶培养物,产量比野生型大肠杆菌提高了10倍以上(Michou等人,2019年)。然而,根据用户的特殊喜好和需要,也可以使用其他MP净化方法。


12.  通过SDS-PAGE分析和考马斯染色评价MP纯度。


注:对于高浓度MP组分,我们建议在非常低的恒流下运行凝胶,例如12-13 mA。


 


食谱


 


1.     鲁里亚贝塔尼肉汤(磅)


10 g/L胰蛋白酶


5g/L酵母抽提物


10 g/L氯化钠


用5 N NaOH将pH调至7.0


高压灭菌器,在15 psi下灭菌20分钟


2.     抗生素


a、 40 mg/ml氯霉素,含99.8%乙醇,-20°C保存


b、 100 mg/ml氨苄西林溶于MilliQ水中,使用0.22μm注射器过滤器对溶液进行消毒,并在-20°C下储存


3.     蛋白质生产诱导因子


a、 20%w/v L(+)-阿拉伯糖溶于MilliQ水中,使用0.22μm注射器过滤器对溶液进行消毒,并在室温下储存


b、 20 mg/L无水四环素(aTc)溶于99.8%的乙醇中,-20°C保存


4.     裂解缓冲液


300毫米氯化钠


50毫米NaH2PO4


15%甘油


5毫米二硫苏糖醇(使用前立即添加)


0.1 mM PMSF(使用前立即添加)


将pH调至7.5


储存于4°C


5.     冲洗缓冲液


20毫米咪唑


300毫米氯化钠


50毫米NaH2PO4


将pH调至8


添加0.1%w/v DDM(使用前立即添加)


储存于4°C


6.     洗脱液


250毫米咪唑


300毫米氯化钠


50毫米NaH2PO4


将pH调至8


添加0.1%w/v DDM(使用前立即添加)


储存于4°C


7.     十二烷基硫酸钠(SDS)样品缓冲液(6x)


6%w/v十二烷基硫酸钠


300毫米Tris


15%v/v甘油


0.01%w/v溴酚蓝


10%v/vβ-巯基乙醇


将pH调至6.8


8.     Tris缓冲盐水与吐温-20(TBST)


20毫米Tris底座


150毫米氯化钠


将pH调至7.5


0.1%v/v吐温-20


9.     磷酸盐缓冲盐水(PBS)


137毫米氯化钠


2.7毫米氯化钾


10毫米Na2HPO4


1.8毫米KH2PO4


将pH调至7.4


高压灭菌器,在15 psi下灭菌20分钟


10.  DDM增溶缓冲液


裂解缓冲液


2.5%w/v DDM(使用前立即添加)


11.  缓冲区大小排除


这个缓冲区应该根据感兴趣的MP的特性来选择。一些例子包括:


0.05%氯化钠或0.0%氯化钠水溶液或0.0%氯化钠水溶液


 


致谢


 


这项工作得到了以下方面的资助:(i)希腊国家奖学金基金会(Idrima Kratikon Ypotrofion–IKY)奖学金,由合作协议“开发人类潜力”的“通过博士研究加强人类研究潜力”行动提供资金,“教育和终身学习”,2014-2020年(赠款编号:MIS 5000432),由欧洲结构和投资基金(ESIF)和希腊政府共同出资,(ii)欧洲研究理事会(ERC)根据欧盟地平线2020研究和创新计划(“ProMiDis”项目;赠款协议编号819934)。


 


相互竞争的利益


 


GS是大肠杆菌SuptoxD和SuptoxR专利申请的发明人(PCT/EP2017/025168)。作者声明没有其他利益冲突。


 


工具书类


 


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引用:Michou, M., Delivoria, D. C. and Skretas, G. (2020). High-level Production of Recombinant Membrane Proteins Using the Engineered Escherichia coli Strains SuptoxD and SuptoxR. Bio-protocol 10(15): e3710. DOI: 10.21769/BioProtoc.3710.
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