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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

Fig.1: Scheme of construct. The insert, containing the promoter P_Hpall, α-s2-casein and the double fd terminator, was cloned into the pBSMUl1 backbone with different secretion signals - here: SPNprE SPNprE

This composite part (Fig.1) contains the constitutive promoter P_Hpall , expressing α-s2-casein with the secretion signal SPNprE and the fd terminator 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

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.

Fig. 2: 250 ml Erlenmeyer flasks with 30 ml LB with [50 µg/mL] Kanamycin, the culture medium for the transformed B. subtilis strains.

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).

Fig. 3: Sodium dodecyl sulfate polyacrylamide gel electrophoresis of B. subtilis untreated growth media to detect secreted proteins. The SDS-PAGE was run at 200V for 45 minutes and then stained with Coomassie blue overnight. α-s2-casein has a molecular weight of ~27 kDa without and ~30 kDa with the signal peptide. Wildtype= Bacillus subtilis DB430

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 the heterologously expressed α-s2-casein secreted proteins were detectable on a coomassie stained gel at all (Fig. 3). This might be caused since the 30ml culture might not be 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 is fused to 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.

Fig. 4: Western Blot of Bacillus subtilis lysed pellet protein. The Wildtype= Bacillus subtili DB430 shows no significant bands. pBSMUl1-SPNprE+α-s2-casein shows no significant bands but a faint band around 15 kDa.

Fig. 5: Fig. 5: Western Blot of B. subtilis concentrated media fraction containing proteins around and over 30 kDa. Wildtype= Bacillus subtilis DB430. The wildtype shows a faint band around ~25 kDa while pBSMu1+SpNprE+α-s2-casein has a band with a very high intensity at this size, which fits to SpNprE+α-s2-casein.

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 than for α-s1-casein and therefore the protein is secreted more efficiently 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.

Conclusion

α-s2-casein was successfully cloned and expressed in B. subtilis. With the focus on optimizing the expression of the protein of interest, five signal peptides (sslipA, SPNprE, SPPel, SPEpr and SPYurl) were tested out. No proteins were detected in the Coomassie stained growth media (Fig. 3), indicating that further test need to be carried out on concentrated fractions to get clearer results. Western Blot analysis of lysed pellet proteins illustrate no protein of interest in the pellet, but an unspecified signal that might be degraded or cleaved protein (Fig. 4). Western Blot analysis of concentrated media fraction containing proteins >30 kDa illustrates the protein of interest indicating, that the α-s2-casein protein fused to the SPNprE signal peptide is secreted into the growth media (Fig. 5).

In comparison to pBSMUl1-SPNprE+α-s1-casein, where it seems, that the protein of interested is retained in the cells alpha S2 casein seems to be secreted by the same secretion tag. This example shows that an optimal tag needs to be found for each individual protein. Many known B. subtilis secretion signals are available for that purpose.

With SPNprE a suitable secretion tag for α-s2-casein is now already identified. The band at 25 kDa resembles our protein of interest, while the band at 15 kDa might be degraded protein. After 12 hours the cells should have reached the stationary phase and do not produce new protein. The produced protein could be degraded after that time point. In further experiments the induction time should be decreased to see if the unspecific band at 15 kDa is decreased and the specific protein band is increased.

References

[1]: Simonen, Marjo, and Ilkka Palva. "Protein secretion in Bacillus species." Microbiology and Molecular Biology Reviews 57.1 (1993): 109-137.

[2]: Sewalt, Vincent, et al. "The Generally Recognized as Safe (GRAS) process for industrial microbial enzymes." Industrial Biotechnology 12.5 (2016): 295-302.

[3]: IFBC (International Food Biotechnology Council). "Chapter 4: Safety Evaluation of Foods and Food Ingredients Derived from Microorganisms in Biotechnologies and Food: Assuring the Safety of Foods Produced by Genetic Modification." Reg. Toxicol. Pharmacol. 12 (1990): S1-S196.

[4]: Food and Drug Administration. "Carbohydrase and protease enzyme preparations derived from Bacillus subtilis or Bacillus amyloliquefaciens: Affirmation of GRAS Status as direct food ingredients. 64 Fed." Reg. 19887 19895 (1999).

[5]: Petsch, Dagmar, and Friedrich Birger Anspach. "Endotoxin removal from protein solutions." Journal of biotechnology 76.2-3 (2000): 97-119.