Editing Part:BBa I746916

Characterization: Freiburg 2019

Group: Freiburg 2019
Author: Fabian Bäzner
Summary: We demonstrated the non-peptide guided auto-secretion of sfGFP (BBa_I746916) into the medium. sfGFP can thereby be used as a carrier for proteins linked to its carboxyl end which we analyzed with different induction conditions to reach optimal secretion.


Documentation:

Secretion of proteins can have various benefits. The secretory production of recombinant proteins leads to simpler purification, avoidance of protease attack and a better chance of correct protein folding1. Due to its fluorescent properties, sfGFP represents a secretion tag for easy extracellular signal validation. To demonstrate the auto-secretion of sfGFP described by Zhang et al.2 we designed two constructs, each containing the Npu DnE C-Intein (BBa_K1362101) with Thioredoxin as an insert (Figure 1). In one construct, the C-intein was fused to the carboxyl end of sfGFP.

Fig. 1: constructs for sfGFP secretion


Expression system:

Backbone: pET302
Promoter: T7 promoter
Terminator: T7 terminator
RBS: T7 RBS (BBa_K1362090)
E.coli strain: BL21(DE3)



We induced the sfGFP-CInt-TRX construct in 20 mL E. coli cultures under different temperatures (37, 30, 25°C), IPTG concentrations (1, 0.5, 0.1 mM, ctrl.), media (LB-medium, auto-induction medium) and took samples of each culture after 16, 24 or 48 hours. The samples were subsequently centrifuged at 21000 xg and the fluorescence of the supernatant was measured with a plate reader (excitation: 495 nm; emission: 515 nm) (Figure 2).


Fig. 2: Fluorescence of the medium after different inductions


The induction with 0.1 mM IPTG at 37°C in LB-medium showed the highest relative fluorescence. This condition was used for a 50 mL culture and the secretion was analyzed over time. We measured the OD600 and obtained samples of the culture every 2 hours. Samples were centrifuged at 21000 xg and the fluorescence of the supernatant was measured with the plate reader (excitation: 495 nm; emission: 515 nm)(Figure 3).


Fig. 3: Fluorescence of the medium and OD600 of the induced and uninduced 50 mL culture containing E. coli BL21(DE3) with the sfGFP-CInt-TRX construct.


After 6 hours, the fluorescence in the medium increases, while the cells are still in the growth phase. These results were confirmed by Western Blot analyses of the supernatant, using an antibody against TRX (Figure 4). Expression of CInt-TRX in another 50 mL culture of E. coli BL21(DE3) under the same conditions did not show secretion of TRX (figure 4, 8 h / 10 h). This indicates the increase of fluorescence in the medium is based on the secretion of sfGFP-CInt-TRX and not on lysis of cells and sfGFP is carrying CInt-TRX into the medium.


Fig. 4: Western Blot of samples from different timepoints from the medium of cultures con- taining BL21(DE3) expressing CInt-TRX or sfGFP-CInt-TRX, all samples adjusted to an OD600 of 4, stained against TRX


Altogether we demonstrated the auto-secretion of sfGFP, analyzed different conditions for the secretion and measured the secretion over time.


References:

[1] Choi J. H. et al., Secretory and extracellular production of recombinant proteins using Escherichia coli (2004). Appl. Microbiol. Biotechnol. 64, 625–635

[2] Zhang Z. et al., Non-peptide guided auto-secretion of recombinant proteins by superfolder green fluorescent protein in Escherichia coli (2017). Scientific Reports. 7, 6990