Part:BBa_J36841

Streptavidin, wild-type core + His6 tag (no start codon)

This part codes for the core part of wild-type streptavidin protein, without the start codon. Streptavidin protein binds strongly to the biotin molecule, and can be used to bind biotinylated nucleic acids or peptides.

There is a histidine tag added at the end of the coding sequence right before the stop codons. You can probe for this protein with an anti-his(C-term) antibody.

It was used as part of a fusion protein with lipoprotein signal peptide and outer membrane protein A transmembrane domains, to express streptavidin on the outer surface of E. coli.

Overview

Methyl-binding proteins (MBD) bind to a specific dsDNA sequence. Streptavidin (SA) is a biotin-binding protein from Streptomyces avidinii. Each SA can bind up to 4 molecules of biotin. The MBD-SA fusion protein we constructed can bind 4 dsDNA sequences. MBDs are trans-acting factors that bind to methylated DNA and can specifically recognize and bind to methylated CpG dinucleotides. MBD has a conserved DNA-binding motif that binds to methylated CpG dinucleotides. Since SA has no glycosylation modification, it has better engineering properties. In order to prepare hydrogels more efficiently, our team improved the existing part (BBa_J36841)， and constructed a fusion protein between MBD and SA to obtain a quadrivalent MBD-SA fusion protein.

Biology

At present, the Methyl-CpG-binding domain (MBD) protein family is known to include 11 proteins, all of which contain a highly conserved protein domain, namely the MBD domain, which has about 70 residues^[1]. MBD protein can act as a transcriptional repressor and participate in a series of transcription processes such as DNA methylation, histone modification and chromatin organization^[2]. In addition to the ability to bind a single symmetrical methylated CpG dinucleotide, the MBD protein family also contains other different domains. Therefore, different members of the MBD protein family exhibit different properties and have different degrees of selective binding to methylated DNA, and some members even bind to non-cpg methylated DNA sites.

Modeled 3D-Structure of the Streptavidin Monomer

Modeled 3D-Structure of the Streptavidin Streptavidin

Modeled 3D-Structure of MBD

Usage

MBD is fused to the N terminus of SA, and one molecule of fusion protein can bind to the partial dsDNA region composed of ssDNA repeats amplified by four RCA reactions (Fig.1) to assemble the DNA nanosponge. The basic part was obtained and the constructed plasmid was transformed into Escherichia coli to verify its expression. Positive clones were cultured. For MBD, fusion with SA does not affect its ability to bind dsDNA, and at the same time, the formation of fusion protein with SA makes four MBDS appear on a fusion protein at the same time. MBD-SA fusion protein can self-assemble into DNA nanosponge after mixing with RCA amplification products. This shows that we can prepare hydrogels efficiently, rapidly, and in large quantities by MBD-SA fusion protein and RCA amplification reaction, which also allows us to bypass the step of modifying ssDNA with biotin, further reducing the cost of hydrogel preparation. Moreover, we can also control the crosslinking degree of the DNA nanosponge by changing the proportion of MBD-SA fusion protein. In addition, MBD and SA have good biological stability and can maintain the original structure in the freeze-dried state, and MBD has considerable dsDNA binding ability in the freeze-dried state. This allows our DNA nanosponge to be stored in the freeze-dried state for a longer time and maintain good biological activity. We also performed experimental detection of the binding activity of MBD-SA fusion protein, which provides more data support for more potential applications.

We designed a nucleic acid nanosponge prepared by RCA to encapsulate the follow-up protein, and this sequence is the primer required for RCA. For circular template, add this single primer complementary to the template, and after hybridization between the primer and the template, roll loop amplification can be started. The synthetic product is repetitive linear single stranded DNA sequence, and the process is linear amplification.

SCHEM 1. DNA -protein hybrid hydrogel based on RCA and MBD-SA

Results

1. Expression of recombinant protein

By SDS-PAGE electrophoresis analysis, as shown in Fig.1: from left to right, 1-9 samples were added to each band: Marker, uninduced, 20℃ total protein, 20℃ supernatant, 25℃ total protein, 25℃ supernatant, 37℃ total protein, 37℃ supernatant, and 15℃ supernatant. SA-MBD was only expressed in the total protein induced by 37℃.

Fig. 1 SDS-PAGE analysis of recombinant protein expression productsLane 1: Marker; Lane 2: not induced; Lane 3:20 ℃ total protein; Lane 4:20 ℃ supernatant; Lane 5:25 ℃ total protein; Lane 6:25 ℃ supernatant; Lane 7:37 ℃ total protein; Lane 8:37 ℃ supernatant; Lane 9:15 ℃ supernatant

2. Identification

SA was verified by Western blot

Sample preparation and electrophoresis:

We prepared 8% separation glue and the matching concentration glue, and the sample volume of each well was 10 μL, and the mixture was evenly mixed (5×loading Buffer 2 μL+ protein sample 8 μL). After water bath, 10 μL sample and 3 μL Marker were added to the sample well. Electrophoresis at 80 V for 30 min was followed by electrophoresis at 120 V for 90 min.

Film transfer:

According to the molecular weight of target protein,we removed a small adhesive strip and the glass plate was gently lifted off under the instructions of Marker. The glue was transferred to the pre-cut 4 layers of filtrate and the mold was turned in a 200 mA ice bath for 1.5 h.

Closed:

The cells were washed three times with TBST buffer for 10 min each time, and added 10 mL blocking solution and shaken at 37 ° C for 10 min. The cells were washed three times for 10 min each using TBST buffer.

Incubation and development:

After HRP-biotin adding, the cells were incubated for 1 h. The cells were washed 3 times with TBST buffer for 10 min each time. The developer was evenly dropped on the surface of the film, and the film was kept away from light for 1 min. Put into the second grid of the instrument for exposure.

As shown in Fig.2, MBD-BCCP as a control group could not show a band when interacting with HRP-biotin. SA-MBD can show a band on ECL by interacting with HRP-biotin. The target protein was confirmed to contain streptavidin (SA).

Fig. 2 Western blot identification of streptavidin

3. Purification and identification of recombinant proteins

The purified recombinant protein was analyzed by SDS-PAGE electrophoresis, as shown in Fig. 3: there was only SA-MBD band between the 25-35 KDa band, and no other bands, that is, other miscellaneous proteins, indicating that the purification effect was good.

Fig. 3 SDS-PAGE identification of purified recombinant proteins

4. Binding Capability Verification

Microthermophoresis (MST)

As shown in Fig. 4/5/6/7 below, the Kd values of MBD and MBD-SA for methylated DNA (M-DAN) and unmethylated DNA (U-DNA), respectively, can be seen from Table 1: The Kd value of MBD-SA to M-DNA (0.19045) was lower than that of MBD to M-DNA (1.4406), which indicated that the affinity of MBD-SA to methylated DNA was higher than that of MBD to methylated DNA. This means that MBD-SA will have higher sensitivity and accuracy than MBD in identifying methylated DNA.

Table. 1 Results of micro-thermophoresis

Fig. 4 Affinity of MBD and U-DNA (Kd=13.341μM)

Fig. 5 Affinity of MBD and M-DNA (Kd=1.4406μM)

Fig. 6 Affinity of MBD-SA and U-DNA (Kd=6.9985μM)

Fig. 7 Affinity of MBD-SA and M-DNA (Kd=0.19045μM)

5. RCA product validation

RCA is a nucleic acid amplification technology established by referring to the rolling circle replication of DNA molecules of circular pathogenic microorganisms in environment. It is a DNA amplification technology that occurs at a constant temperature. In the RCA reaction, when there is a circular DNA template and polymerase, by the action of DNA polymerase, the circular DNA is used as the template for replication, and finally a single strand DNA is formed, with a repeat sequence complementary to the circular DNA template.In order to verify that the RCA reaction we designed can amplify the bands we wanted to obtain, we performed agarose gel electrophoresis on the reaction products.

Fig. 1 Agarose gel electrophoresis of different products of the whole RCA process. Lane 1: Linear DNA; Lane 2 : DNA with stem-loop; Lane 3, Lane4, Lane5: DNA nanosponge.

6. Direct observation

In order to verify that MBD-SA fusion protein can construct DNA nanosponge more intuitively, we used scanning electron microscopy (SEM) to directly observe the DNA nanosponge we prepared. We mixed MBD-SA fusion protein with RCA amplification products and incubated them at 37℃ for more than 6 h, flocculent material appeared. The cells were centrifuged at 10000×g for 1 min to remove the supernatant. The cells were washed three times with 100 μL of ddH2O. We obtained a DNA nanosponge, as shown in Fig.8. We then lyophilized the samples and directly observed our hydrogels using SEM. (Fig.9)

Fig. 8 DNA nanosponge

Fig. 9 DNA nanosponge under SEM

References

[1] Jeltsch A, Broche J, Lungu C, et al. Biotechnological Applications of MBD Domain Proteins for DNA Methylation Analysis. Journal of Molecular Biology432, 1816-1823(2020)

[2] Baubec T, Ivanek R, Lienert F, et al. Methylation-Dependent and -Independent Genomic Targeting Principles of the MBD Protein Family. Cell153,480-492(2013)

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 40
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 82
  Illegal AgeI site found at 133
  
• 1000
  COMPATIBLE WITH RFC[1000]