Part:BBa_K4661015
Engineered HFBI-sfGFP fusion
Engineered HFBI-sfGFP fusion, linked by a flexible linker (SGGSGGS). All 8 cysteines residues from the natural sequence of HFBI in Trichoderma reesei were replaced with serines with a view towards improving its expression and minimizing aggregation in oxidizing conditions.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 400
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
Hydrophobins are small fungal proteins with demonstrated emulsion formation properties (1). In recent years, they have gained significant attention in industrial applications for their physical properties. Useful in processes such as drug delivery in the pharmaceutical industry, the food industry, or for the creation of cleaning products, hydrophobins still remain challenging to produce at scale in many heterologous systems such as E. coli due to improper folding in the E. coli cytoplasm, which lacks the disulfide isomerase needed to correctly fold sequences with four disulfide bridges (1,2). Moreover, these small proteins are usually formed in inclusion bodies, and their isolation requires extensive denaturation/renaturation steps, which can compromise the protein stability and function. We pursued a rational design strategy to enable high level expression of hydrophobins in a E. coli BL21 chassis.
Based on previous reports that targeted mutations to hydrophobin’s cysteine residues to improve the production and secretion of hydrophobins (3), we designed an HFBI hydrohphobin sequence from Trichoderma reesei in which we substituted all cysteine residues with serines. We created a fusion between the modified HFBI sequence, via a short linker coding for the amino acids SGGSGGS, to a His-tagged sfGFP reporter. We placed this composite part within a pET29 vector, under the transcriptional control of a tet promoter, inducible with anhydrotetracycline.
Use in the Community
This composite part is useful to the community, as it provides a way to easily track hydrophobin expression through the sfGFP fusion, in a series of experiments such as fluorescence microscopy or flow cytometry. Moreover, we showcase a a strategy for improving the protein production of HFBI by the removal of disulfide bridges between cysteines in the endogenous protein sequence from Trichoderma reesei. This strategy paves the way for improving production performance in an E. coli chassis, without compromising on the physical properties of the proteins.
Characterization
The pET29 expression plasmid was transformed into BL21 cells that were then isolated as single colonies. Protein expression was induced in shaking flask cultures generated from overnight liquid cultures of BL21 cells. (Note: Instead of the proper aTc inducer, our team mistakenly used IPTG in induction of expression for this part, so all the expression levels monitored below are a result of leaky expression from the promoter. We anticipate that in the presence of the proper inducer, the part would exhibit even higher performance).
Purification
As the HFBI-GFP fusion also harboured a His-tag, the protein could be purified using affinity chromatography with a Ni-NTA resin. The result of the expression and purification experiment was assessed by SDS-PAGE gel electrophoresis.
Approximate molecular weight mutated HFBI-sfGFP fusion: 35.87 kDA
Approximate molecular weight mutated sfGFP-6His fusion: 27.85 kDA
The gel image bellow confirms the expression of the HFBI-sfGFP fusion (top band on HFBI mutant lane marked with a blue arrow), as well as potential cleavage of the GFP sequence. The second and third lanes were loaded with purified protein coding for sfGFP fusions of the MBSP1 protein, as well as the native (not engineered) version of HFBI, respectively. An alternative hypothesis to that of the spontaneous in vivo cleavage is that the bands highlighted in yellow on the gel do indeed represent the entire hydrophobin-sfGFP fusion, but migrate slightly differently than expected on the SDS-PAGE gel. More experiments should be performed to validate the identity of the bands observed on the gel.
It is also apparent that the mutations introduced in the HFBI sequence, which differentiate this part from BBa_K4661013 (labeled as HFBI on the gel) significantly increased the capacity of the cells to produce the hydrophobin. As a consequence of the C->S engineering, both the free GFP and GFP-conjugated HFBI bands increased significantly in intensity. As proper controls were not included in this measurement, it is necessary to perform future measurements with multiple replicates and a normalized input of material. Alternatively, quantitation of the GFP signal as a proxy for recombinant protein production could be achieved using flow cytometry.
Live-cell Time-lapse Imaging
We also investigated the protein expression system in more depth. After noticing that our cells carrying the mutated HFBI-sfGFP plasmid were intensely fluorescent in the absence of the inducer, we set out to further investigate the morphological defects that cells expressing hydrophobin-GFP fusions could exhibit. To do so, we partnered with a local lab at KU Leuven to run a live-cell time-lapse imaging experiment to follow the fluorescence levels of BL21 cells , in the presence and absence of the inducer. A representative population of cells can be tracked in the images below, 1 hour after induction with IPTG, for 20 distinct time points that span more than 6 hours. The experiment showed BL21 cells exhibiting bright green fluorescence, even in the absence of the inducer. Fluorescence was observed homogeneously throughout the cells, in contrast to the expected observation of accumulation in inclusion bodies in the case of natural hydrophobin sequences. Moreover, as cells were observed dividing, slight morphological defects were observed, such as abnormal elongation or asymmetrical aggregate formation at the poles of the cells. However, these defects were less pronounced than in the case of part BBa_K4661013, where the disulfide bridges between cysteines were maintained.
Future Improvement to the Part
High levels of fluorescence were observed in the absence of protein induction in BL21 cells transformed with a pET29 plasmid carrying part BBa K4661015. While not a direct property of the part itself, the leakiness of the system is a characteristic we wish to improve upon in the future, by using more tightly regulated promoter sequences from which the part can be expressed. Upon careful investigation, our team discovered the wrong inducer was used to trigger protein expression. Therefore, IPTG was used instead of aTc. This observation highlights the need for improvement of a leaky system, which gave rise to relatively high levels of expression in the absence of a proper inducer. Moreover, this mistake also provides the possibility of much higher potential levels of protein expressed when the proper inducer is used in our system. Since too strong of an expression induction could lead to toxic effects, future improvement of the part also needs to focus on deriving a tightly controllable expression system.
For better performance in the future, we are considering engineering the part with an additional N-terminal signal peptide, to target the fusion protein for secretion through the Sec pathway. This would potentially alleviate the accumulation phenomenon observed in cells producing hydrophobins, especeially in those where cysteine mutaitons were not engineered.
Our team derived a script to generate mutations in protein sequences to lower the isoelectric point of proteins. The computational workflow can be run to generate amino acid substitutions in the sequence of the HFBI hydrophobin to find other mutable sites that can improve upon the properties of hydrophobins, without compromising on the essential emulsion formation trait for which we express these proteins.
Source
GenBank accession number for the HFBI sequence that was mutated to generate part : 2FZ6_A
GenBank accession number for sfGFP: MN146014.1
References
1. Cui, L., Cheng, C., Qiu, Y., Jiang, T. & He, B. Excretory overexpression of hydrophobins as multifunctional biosurfactants in E. coli. Int. J. Biol. Macromol. 165, 1296–1302 (2020).
2. Khalesi, M., Gebruers, K. & Derdelinckx, G. Recent Advances in Fungal Hydrophobin Towards Using in Industry. Protein J. 34, 243–255 (2015).
3. Cheng, Y. et al. Soluble hydrophobin mutants produced in Escherichia coli can self-assemble at various interfaces. J. Colloid Interface Sci. 573, 384–395 (2020).
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