Team:Hong Kong HKU/proteinexpression

Protein Expression

Four plasmids, (1) pET28a, (2) pETE, (3) pSB1C3, (4) pSB1C3-BBa_K311004, were electroporated separately into competent BL21(DE3) cells. We then inoculated the cells in 10ml of LB medium supplemented with their corresponding antibiotics (pET28a system with Kanamycin; pSB1C3 system with Chloramphenicol) at 37°C for 4-8 hours until OD ~0.6-0.8, and cooled the cells down on ice. 1ml of sample is withdrawn and kept at 4°C for next day's SDS-PAGE analysis.

Protein expression was carried out as follows using two methods. (1) IPTG was added to a final concentration of 0.2mM after 4 hours of growth at 37°C, 200rpm. The induction was carried out overnight at room temperature (25°C) with shaking at 200rpm. (2) Cells are transferred to a 30°C incubator without any IPTG and was incubated overnight with shaking at 200rpm, to allow leaky expression of the promoters.

The next day whole cells were boiled down with SDS loading dye supplemented with β-mercaptoethanol for 10 minutes, and samples run on an 18% SDS-PAGE gel for analysis.

Samples (for each of the plasmid constructs) analyzed on an SDS-PAGE were as follows: (1) pre-induction/expression: 1ml sample of was withdrawn after 4 hours of incubation at 37°C, cooled down to 4°C and kept for next day's analysis
(2) post-induction 3 hours: 3-hours after the two methods of protein expression has started another 1ml of sample was withdrawn and kept at 4°C overnight for next day's analysis
(3) complete induction: overnight cells were cooled down and an appropriate volume of cells withdrawn for SDS-PAGE. Cells concentrations were normalized according to their OD, and boiled with SDS-PAGE supplemented with 1% β-mercaptoethanol for 10min. Finally, the boiled samples were run on a 8% stacking gel/18% separating gel for analysis. The electrophoresis was carried out for 2h at 120V. The gel was then recovered and stained with Coomassie Blue stain for 1h and destaind overnight with destaining solution (water:methanol:glacial acetic acid 50:40:10).

Interestingly, the results gave extra information than expected. Firstly, the difference in size between EutS and the His6-tagged EutS is readily apparent in the strongest band, and it also shows that both EutS and His6-EutS are well-expressed. Smearing of the bands suspected to be EutS (11.6kDa), His6-EutS (13.8kDa) and EutM (9.8 kDa) showed some smearing that is typical of proteins rich in hydrophobic residues. The EutN (10.4kDa) band was difficult to discern from that of EutM in both cases, as in the literature. The band related to EutK (17.5kDa) was relatively vague in both pETE and BBa_K311004, but is also consistent with that observed in the literatures which showed similar results. EutL (22.7kDa) was also clearly expressed.

Interestingly, different conditions seems to have an impact on different system. Comparing pETE with BBa_K311004, the only differences are (1) EutS was under T7 promoter in pETE rather than under Plac in BBa_K311004, in both cases, the spacing regions between the promoter and putative transcriptional start site, and that between the consensus RBS and start codon are both optimal and the same. (2) EutS in pETE was His6-tagged together with a thrombin site before fusing to EutS, while that of BBa_K311004 is not. From our data, it seems that the expression for His6-EutS from pETE is better with 0.2mM IPTG room temperature overnight induction, while that of BBa_K311004 is better with overnight 30°C incubation. On the other hand, EutL is better expressed with overnight 30°C incubation than for using IPTG, although one could argue that these samples were normalized according to the cultures’ OD, so that the protein maybe under-represented in terms of percentage of global protein concentration and thus also for band intensity, such conclusion was invalid as the result from BBa_K311004 clearly shows such under-representation was absent. Furthermore, the regulation of the T7 promoter was rather tight, as indicated by the fact that very little His6-EutS was produced if IPTG was not added in 30°C incubation. Intriguingly, there is one ~26-28kDa band visible in both cases that does not match with any calculated sizes of proteins expressed by the plasmids compared with the controls, indicating that the presence of BMC might affect the intrinsic protein expression of BL21(DE3) cells.


*Pre-I: Pre-induction or expression; 3h Post-I: 3 hours post-induction with 0.2mM IPTG; ON-I: overnight induction with 0.2mM IPTG; ON-30°C: overnight incubation at 30°C without IPTG. Calculated sizes of proteins for reference: EutS 11.6kDa, His6-EutS 13.84kDa, EutM 9.8kDa, EutN 10.4kDa, EutL 22.7kDa, EutK 17.5kDa