Part:BBa_K2924052
Hpall + SPNprE + alpha s2 + fd terminator
PHpaII with RBS expressing SPNprE+ α-s2-casein + 6xHis-tag, terminated by the fd Terminator in Bacillus subtilis.
Usage and Biology
This composite part (Fig.1) contains the constitutive promoter PHpall (BBa_K2924043), expressing α-s2-casein (BBa_K2924027) with the secretion signal SPNprE (BBa_K2924047) and the fd terminator (BBa_K2924044) for expression and secretion of the protein in Bacillus subtilis.
B. subtilis is a frequently used expression system for secreting proteins, which can avoid some common problems with intracellular over expressions like low expression rates, improper protein-folding, formation of inclusion bodies or product toxicity. The secretion is achieved by secretion signals, which are fused to proteins, leading to an export of those tagged proteins by different mechanism, while the secretion tag is cleaved of in many cases after successful secretion. The organism has been widely described and examined; its genome has been fully sequenced and all important genes and metabolic pathways are known. The fundamental architecture of B. subtilis cell wall can ease protein secretion pathways and allow the organism to secrete high levels of extracellular proteins directly into the medium1. For food production, the gram positive bacterium is highly favored, given the fact that it is examined as a GRAS organism (generally recognized as safe)2,3,4. Furthermore, compared to other organisms, it does not produce endotoxins that are wished to be removed of the final product5. Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1124
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 554
Characterization
For expression and secretion of α-s2-casein the gene was cloned into the high-copy pBSMUl1-SPNprE plasmid, N-terminally fused to the signal peptide SPNprE and C-terminally fused to a 6xHis-tag for easier purification and immunodetection.
A single positive transformant of the pBSMUl1-SPNprE+alpha-s2-casein plasmid was inoculated into 5 ml LB medium containing 50 µg/ mL Kanamycin and incubated at 37°C, 250 rpm for the next 24 hours. After 24 hours, 29 ml LB Medium were inoculated with 1 ml overnight culture and incubated at 37°C, 250 rpm for 48 hours. We used erlenmeyer flasks with a volume of 250 ml to ensure sufficient aeration (Fig. 2). Figure 2: 250 ml Erlenmeyer flasks with 30 ml LB with [50 µg/mL] Kanamycin, the culture medium for the transformed Bacillus strains.
Afterwards, the cells were harvested by centrifugation at 8000 x g for 20 min at 4°C. Culture supernatant and cell pellets were kept both on ice. The cell pellet was lysed with Bacterial Protein Extraction Reagent (Thermofisher) and incubated for 60 minutes at room temperature. The lysate was centrifuged at 15,000 × g for 5 minutes to separate soluble proteins from the insoluble proteins. To separate proteins with molecular weights of >30 kDa and <30 kDa in the growth media - with α-s2-casein having a molecular weight of ~27 kDa without and ~30 kDa with the signal peptide- Amicon ultra centrifugation columns, with a cutoff of 30 kDa were used. The protein of interest should be located in the concentrated fraction together with all proteins >30 kDa since it is close to the column cutoff and therefore should be retained in the column and not be in the flowthrough. The flowthrough and concentrated protein fractions were further treated with acetone to precipitate the proteins. For that, 4 times the volume -20°C acetone was added to the extract and vortexed. The sample was incubated at -20°C for 2 hours. Afterwards, it was centrifuged at 14000 x g for 4 min at 4°C and the supernatant was discarded. The pellet was washed twice with a 4:1 acetone/water mixture at -20°C until the pellet was well broken up. Between each washing step the solution was centrifuged at 14000 x g at 4°C for 10 min. After the final centrifugation, the solution was incubated at 40°C until approximately 80% of the liquid was evaporated. The pellet was resuspended in the remaining liquid, mixed with SDS loading dye and analysed by SDS-PAGE.
Neither B. subtilis native nor heterologous expressed α-s2-casein secreted proteins were detectable on an coomassie stained gel at all (Fig. 3). This might be caused since the 30ml culture might be not concentrated high enough to see the proteins. To detect smaller amounts of protein western blots on different B. subtilis fractions were executed. α-s1-casein has a molecular weight of ~27 kDa without the secretion signal and ~30 kDa with the secretion peptide. No bands could be detected in the SDS-PAGE gel for WT and pBSMUl1-SPNprE+α-s1-casein. The expressed protein athands a 6xHis tag. For detection, a anti-his antibody is used and further treated on western blot .
For western blot preparation the membrane was first presoaked in 100% ethanol, then in blotting buffer. The blotting sandwich was created and run on 350-500 mA for 1 hour. Afterwards, the membrane was blocked in 1x TBS-BSA and incubated overnight at 4°C on a rocking incubator at 20 rpm. The membrane was washed with a solution containing the 6x-HIS Tag Monoclonal antibody and incubated for 1 hour. After washing of unbound primary antibody with 1x TBST the secondary antibody (mouse) at 1 : 5000 dilution was added and the membrane was incubated with it at room temperature for 1 hour. The membrane was washed again with 1x TBST. Immunodetection was carried out with a chemiluminescence detection kit (Thermo Fisher) to detect the activity of the horseradish peroxidase, which is bound to the secondary antibody.
α-s2-casein has a molecular weight of ~27 kDa without and ~30 kDa with the signal peptide. The western blot of the lysed pellet shows no strong band for the wildtype, while showing a faint band for pBSMUl1-SPNprE+α-s2-casein at 15 kDa (Fig. 4), however there is no strong band at the expected sizes at ~27 or 30 kDa as there is for pBSMUl1-SPNprE+α-s1-casein. Possible reasons for that might be that the protein was digested or cleaved. Furthermore, another protein bearing the same or similar epitope might be detected differently by the antibody. Another, more favorable, explanation could be, that the secretion tag works more efficient for α-s2-casein then for α-s1-casein and therefore the protein is secreted more efficient to the medium. To prove, that our protein of interest is secreted into the growth medium another western blot was executed with the concentrated fraction which should contain proteins over 30 kDa (Fig. 5). This fraction and not the fraction below 30 kDA was used, since the cutoff is close to the molecular size of the protein of interest and it therefore should be retained in this fraction.
The western blot of the concentrated protein fraction containing proteins >30 kDa for SpNprE+α-s2-casein shows a band at ~25 kDa, while the wildtype shows no band, indicating, that the protein is secreted into the cell medium and is contained in the concentrated fraction (Fig. 5).
With our protein of interest , being ~27 kDa without and ~30 kDa with the signal peptide, it is highly possible that during the filter step proteins with this particular size did not flow through the column, since the cutoff was not chosen properly. For further experiments it should be chosen as: (Protein size / 2). Also it can be because the spinning time was too short for the proteins of interest to flow through the column.
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