Part:BBa_K5376002
Core streptavidin
Core streptavidin involves truncating the full-length streptavidin (SA) by cleaving it at positions 10-12 at the N-terminus and 19-21 at the C-terminus, reducing its length from 159 amino acids to 127 amino acids. The water solubility of core streptavidin is greatly improved, reducing abnormal aggregation in cells and making it more suitable for protein fusion. In our project, we fused RFP tags into the C-terminus of SA and cSA and measured their fluorescence values for analysis. It was found that the cSA and RFP fusion protein had high expression levels and fluorescence values in Escherichia coli Nissle 1917, while the SA and RFP fusion protein produced almost no fluorescence. This result was also confirmed by laser confocal microscopy. Table of Contents:
1. Overview
2. Design and Results
2.1 Full-length Streptavidin Expression
2.2 Full-length Streptavidin Fusion with mRFP1 Expression
2.3 Core Streptavidin Fusion with mRFP1 Expression
2.4 Lactococcus lactis Surface Display of Core Streptavidin
2.5 Cell Display Technology of Streptavidin Fusion with MBD93
3. Implementation
Streptavidin (SA) is a protein secreted by Streptomyces, characterized by its high-affinity binding to biotin. The molecular weight of streptavidin is approximately 66.5 kDa, composed of four identical subunits forming a tetramer structure, with each subunit capable of binding to one molecule of biotin. Thus, the entire tetramer can bind four molecules of biotin. The binding between streptavidin and biotin is extremely strong, with a dissociation constant of about 10^-14 mol/L. This intense interaction makes streptavidin widely used in biochemistry research and medical diagnostics, such as immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and fluorescence in situ hybridization (FISH).
The natural strong binding affinity, in our team’s view, has many potential applications. On this page, we will describe how we construct streptavidin and expand its applications in biology and medicine.
In our project, we have developed various potential applications based on streptavidin:
• We constructed a fusion protein of mucin-binding protein and streptavidin, which can be excreted by EcN. After biotinylation of the surface of EcN, MBD93 with streptavidin can be successfully displayed on the surface of engineered EcN, enhancing its colonization ability in the gut.
• We constructed a fusion protein of mRFP1 and streptavidin and tested the results, which will be helpful for bio-markers and other applications.
• We used surface technology in Lactococcus lactis to display core streptavidin and the fusion protein of core streptavidin with mRFP1. Lactococcus lactis displaying core streptavidin can bind to biotinylated cell surfaces, and surface display technology will further expand the applications of streptavidin.
To verify whether streptavidin (SA) can be expressed in EcN, we used the strong constitutive promoter J23119 to express SA in EcN. After 48 hours of cultivation, proteins were extracted and subjected to SDS-PAGE, where the protein band of SA should be at 18.9 kDa.
Figure1 SDS-PAGE of J23119 Protein
No obvious band was observed near 18.9 kDa in the gel, suggesting that full-length streptavidin is difficult to express.
We replaced the constitutive J23119 promoter with an inducible T7 promoter and added an RFP tag at the C-terminus of SA to construct the plasmid pETDuet-1-T7-SA-RFP for observation and characterization.
The results showed that the fluorescence values of EcN strains expressing SA-RFP before and after induction were both low, and the BL21 (DE3) strain expressing SA-RFP had a weak fluorescence after induction.
SA-RFP protein may be difficult to express in EcN, or there may be structural abnormalities. Literature analysis suggests that SA tends to aggregate and has poor water solubility, which may bring certain cytotoxicity while also being easily degraded by proteases within the cell. The RFP-tagged SA may be degraded by intracellular proteases, resulting in low fluorescence values.
Some researchers have truncated SA to form core streptavidin (cSA), which can greatly improve its solubility and reduce its aggregation, and we plan to adopt the same strategy.
We broke the full-length SA at positions 10-12 on the N-terminus and 19-21 on the C-terminus, reducing the 159 amino acids of SA to 127, and re-designed and constructed the plasmids pETDuet-1-T7-cSA and pETDuet-1-T7-cSA-RFP.
We cultured the obtained strains in test tubes containing 20 ml of LB for 26 hours, measuring their OD and fluorescence values every 2 hours.
Figure3 OD values (a, b) and fluorescence values (c, d) of EcN and BL21 (DE3) strains expressing cSA-RFP at different times The analysis results showed that the cSA-RFP protein could be normally expressed in both EcN and BL21 (DE3).
This can be applied not only to bio-markers but also to extract proteins for other applications.
To further expand the application scenarios of streptavidin, we constructed the Usp45 signal peptide and 3Lys M motif at the N-terminus of streptavidin to mediate the display of streptavidin on the surface of Lactococcus lactis F44, enabling it to bind to biotinylated surfaces.
Based on core streptavidin, we constructed two vectors: pLEB124-P45-USP45-3LysM-cSA and pLEB124-P45-USP45-3LysM-cSA-mRFP.
We took the culture medium cultured for 48 hours for laser confocal observation, and there was red fluorescence outside the cell wall.
Figure4 Fluorescence confocal photo of Lactococcus lactis F44 displaying cSA-RFP
We need to further verify whether Lactococcus lactis F44 displaying streptavidin on the surface can bind to biotinylated surfaces. We selected Lactococcus lactis F44 displaying cSA and EcN for cultivation. After biotinylating EcN and washing twice with PBS buffer, resuspending in PBS buffer, and mixing and incubating with Lactococcus lactis F44 of the same OD (1OD) washed twice with PBS buffer and resuspended in PBS buffer for three hours, followed by Gram staining observation.
1. Lactococcus lactis displaying cSA and non-biotinylated EcN
Figure5 Gram staining photo of Lactococcus lactis displaying cSA and non-biotinylated EcN aggregation
2. Lactococcus lactis displaying cSA and biotinylated EcN
Figure6 Gram staining photo of Lactococcus lactis displaying SA and non-biotinylated EcN aggregation Lactococcus lactis F44 displaying cSA on the surface can bind to biotinylated surfaces.
In our project, we designed a new cell display technology based on the combination of streptavidin and biotin. We hope to achieve this display technology through the secretion of fusion proteins of streptavidin and target proteins, and the biotinylation of the target strains. We biotinylated the EcN strains expressing cSA-RFP and SA-RFP, further incubated them with the fermentation liquid, and then took 20μL for microscopic observation.
Figure7 Laser confocal photos of EcN strains expressing cSA-RFP before (a, b, c) and after biotinylation (d, e, f) It can be seen from the figure that after biotinylation on the surface of EcN, the red fluorescence in the field of view increased significantly and was more aggregated, indicating that the surface display strategy using streptavidin-biotin is effective.
Figure8 Laser confocal photos of EcN strains expressing SA-RFP before (a) and after biotinylation (b) We also further verified the EcN strains expressing SA-RFP and found that there was almost no red fluorescence in the field of view, indicating that SA-RFP cannot glow correctly, which is consistent with the previous experimental results.
We also tried to display the MBD93 domain on the cell surface. We designed to fuse the full-length SA and the truncated cSA at the N-terminus and C-terminus of MBD93 for measurement.
To verify the display of the MBD93 domain on the cell surface, we inoculated the correctly transformed strains into a conical flask containing 30ml LB and cultured for 24 hours, then biotinylated and took samples to add to the mucin-coated 96-well plate for measurement.
Figure9 The binding effect of cSA-MBD93 to Mucin
The analysis results showed that the strains expressing MBD93-cSA had weak adhesion to mucin before biotinylation, and the adhesion was enhanced after biotinylation, indicating that this surface display strategy is effective, but the structural issues of the protein fused with core streptavidin need to be considered.
3 Implementation
cSA has a wide range of potential applications in the field of bio-detection. We have achieved its expression in Escherichia coli and Lactococcus lactis F44, and explored its application scenarios. Here are some of the main application scenarios:
(1) Cell labeling and tracking: cSA fused with fluorescent proteins can be used for long-term labeling of biotinylated cells. The fluorescent properties of this fusion protein allow for direct detection under a microscope.
(2) Molecular interaction studies: cSA can be used to study interactions between proteins. For example, by observing the co-localization of cSA-RFP labeled proteins within cells, one can infer their interactions.
(3) Bio-detection and analysis: The high-affinity binding characteristics of cSA fused with fluorescent proteins make it suitable for the capture and detection of biomolecules, such as in immunoassays and molecular diagnostics.
(4) Cell surface display technology: We have explored in our project that the use of biotin-streptavidin cell surface display technology is effective.
These applications demonstrate the versatility and flexibility of cSA-RFP as a powerful biological tool in basic research and clinical applications.
Sequence and Features
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