Part:BBa_K2924054
Hpall + SPYurl + alpha s2 + fd terminator
PHpaIIwith RBS expressing SPYurl+ α-s2-casein with a 6xHis-tag, terminated by the fd Terminator in Bacillus subtilis.
Usage and Biology
This composite part (Fig.1) contains the constitutive promoter PHpall, expressing α-s2-casein with the secretion signal SPYurl 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1127
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 557
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 SPYurl and C-terminally fused to a 6xHis-tag for easier purification and immunodetection.
A single positive transformant of the pBSMUl1-SPYurl+α-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).
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.
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.
Due to time issues only the concentrated protein fraction containing proteins around the size and higher than 30 kDa was analysed by western blot (Fig. 3).
The western blot of the concentrated media fraction containing proteins over and around 30 kDa indicates, that some of our target protein is secreted out of the B. subtilis cells, however not as strong as with another signal peptide (SPNprE)
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. 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 SPYurl signal peptide is secreted in low quantities into the growth media (Fig. 5).
In comparison to pBSMUl1-SPNprE+α-s1-casein (BBa_K2924052) the secretion is increased, but in comparison to pBSMUl1-SPNprE+α-s2-casein (BBa_K2924052) the protein secretion is decreased. Many known B. subtilis secretion signals are available for that purpose. With SpYurl we found a signal peptide, that is less suitable than SPNprE for α-s2-casein secretion in B. subtilis.
The band at 25 kDa resembles our protein of interest. 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 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.
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