Difference between revisions of "Part:BBa K2924033"

Latest revision as of 12:14, 21 October 2019

Hpall + SPNprE + α-s1-casein + fd terminator

P_HpaII with RBS expressing SPNprE+ α-s1-casein + 6xHis-tag, terminated by the fd Terminator in Bacillus subtilis.

Usage and Biology

Fig. 1: Scheme of expression construct for B. subtilis. The insert, containing the promoter P_Hpall, the α-s1-casein gene and the double fd terminator, was cloned into the pBSMUl1 backbone with different secretion signals - here: SPNprE

This composite part (Fig. 1) contains the constitutive promoter P_Hpall, expressing α-s1-casein gene with the secretion signal SPNprE and the fd terminator for expression of the protein in Bacillus subtilis.

B. subtilis is a frequently used expression system for secreting proteins, which can avoid some common problems that intra-cellular expression has. Those are: low expression rates, improper protein-folding, formation of inclusion bodies or product toxicity. The secretion is achieved by signal peptides, which are fused to proteins, leading to an export of those tagged proteins by different mechanism, while the signal peptide 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-s1-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+α-s1-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 containing 50 µg/mL Kanamycin were inoculated with 1 ml overnight culture and incubated at 37°C and 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 (Thermo Fisher) 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-s1-casein having a molecular weight of 25,4 kDa without and 28,5 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.

Fig. 3: Sodium dodecyl sulfate polyacrylamide gel electrophoresis of B. subtilis untreatened growth media to detect secreted proteins. The SDS-PAGE was run at 200V for 45 minutes and then stained with Coomassie blue overnight. α-s1-casein has a molecular weight of 25,4 kDa without and 28,5 kDa with the signal peptide. Wildtype= Bacillus subtilis DB430

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-s1-casein secreted proteins were detectable on an commassie 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 25,4 kDa without the secretion signal and 28,5 kDa with the secretion peptide.. No bands could be detected in the SDS-PAGE gel for wildtype 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-s1-casein shows two bands at 15 kDa and ~25 kDa.

Fig. 5: Western Blot of B. subtilis media fraction containing proteins around and over 30 kDa. Wildtype= Bacillus subtilis DB430. Both the wildtype and pBSMu1+α-s1-casein show a faint band around ~25 kDa which have nearly the same intensity.

A western blot of the lysed cell pellet shows no strong bands for the WT, while showing two intense bands for pBSMUl1-SPNprE+α-s1-casein at 15 kDa and ~25 kDa respectively (Fig. 4). The band at 25 kDa corresponds to the expected size of α-s1-casein, while the band at 15 kDa might be degraded protein.

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 show no difference between the wildtype control and the strain expressing SpNpre+α-s1-casein indicating, that the protein is retained in the cells and not secreted into the medium (Fig. 5).

Conclusion

The gene α-s1-casein was successfully cloned and expressed in Bacillus 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 lysed pellet proteins illustrate the protein of interest inside the cells, indicating that the signal peptide SPNprE was suitable for α-s1-casein production (Fig. 4), but not for secretion, since no protein is shown in the growth media (Fig. 3) or in the concentrated media fraction containing proteins >30 kDa (Fig. 5).

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.