Part:BBa_K2881006
Hybrid MBD-HRP
This is an improved part from BBa_K2881003 and BBa_K2881004 working on the same project (check the description pages of them for detailed information). Unlike the two parts, this part fuses a single mouse MBD (mMBD) and a human MBD (hMBD) domain in the fusion protein. Tandem MBD domains are expected to have higher affinity toward methylated CpG site based on the theory of avidity; Hybrid domains were chosen to combine the advantages of two MBD analogs: mMBD domain offers better yield while hMBD domain has higher affinity. Unlike previous two constructs, a Horseradish peroxidase (HRP) is directly fused to the recombinant protein, allowing single step detection and signal amplification.
Biology
First reported in 1998, Methyl-CpG-binding domain proteins are a family of DNA binding proteins specifically recognizing symmetrically methylated CpG sites on DNA. Multiple variants exist in this family including MBDs 1-4 and MeCP2. These proteins all share an analogous Methyl-CpG-binding domain (MBD), which is the functional part to recognize methylated CpG sites (1). MBD proteins have been studied extensively, revealing that many of them are involved in gene expression regulation (2, 3). One important way for cells to control gene expression is to control methylation levels of CpG sites in the promotor regions. CpG sites are dinucleotides in DNA when a cytosine (C) is followed by a guanine (G) in 5’ to 3’ fashion. Many of them are co-incident with gene promotors and could be modified by methyltransferase/demethylase to add/remove methylation (methyl group at 5’ cytosine) (4). Altered methylation status of promotor CpG sites have been shown to involved in multiple cancers (5). MBD proteins serve as the “epigenome reader” to read the methylation status of CpG sites by MBD domain (6); in vivo, their functions such as protein interactions with HDAC1 (7) could then be achieved by other domains.
Design
In our project, we aim to build a general platform to detect and quantify methylation levels of promotor CpG sites in circulating cellular tumor DNAs (ctDNA). MBD domains have been cloned and expressed in the absence of other domains in MBD proteins; research has also demonstrated that MBD domains alone retain their methylated CpG binding affinity in vitro (8). This piece of evidence lays the foundation of our effort to repurpose MBD domain to detect CpG methylation in in vitro assay contexts. In addition, our design of the MBD parts are advised by the following research:
MBD domain from MBD2 protein
MBD proteins not only vary in their functions, but also has significant differences in their MBD domains. Sequences alignment has been reported to demonstrate the variances in the amino acid sequences. Unsurprisingly, MBD domains in different MBD proteins have also been shown to exhibit different affinities toward methylated CpG sites (9). MBD2 protein was shown to have highest affinity in the protein family; among different species human MBD2 protein gives the highest affinity of 5.9nM (10).
Figure 1. Sequence alignment for MBD family proteins
Another key distinction among MBD domains is their sequence specificity. MeCP2, for example, selectively associates with sequence-specific CpG sites when naturally coupled with an A/T rich sequence. Its binding activity is lost when the sequence is deleted. This largely limits its potential for a general assay to characterize CpG sites in different sequences. On the other hand, MBD2 shows little sequence specificity (11). Coupled with the advantage of high affinity MBD2 is the best candidate for our DNA methylation quantification project.
MBD-HRP fusion
Horseradish peroxidase (HRP) is a well characterized and documented enzyme to amplify biomolecules binding activities via its enzymatic reactions. It is most commonly used in ELISA, in which antigen is recognized by antibody conjugated with HRP. The at methylated CpG sites are the "antigens" and MBD domain is the "antibody" encourage us to create this construct for our project. HRP accepts multiple substrates including bi-p,p'-4-hydroxyphenylacetic acid, which could generate fluorescent signals excited at 320nm (13). In our project we chose fluorescence as our detection method since it is compatible with graphene oxide platform (see complement platform), which also relies on fluorescence for signal quantification.
Figure 2. Structure of hm-HRP construct
Characterization
recombinant protein expression and quantification
The constructed plasmids with their sequences confirmed was transformed into E.coli strain BL21 (DE3) for protein expression. A starter culture containing 6mL autoclaved LB broth and 50ug/mL Kanamycin was grown for each construct. The incubation at 37 degree generally took around 3 hours for the culture to become cloudy. Then, 1.5 mL starter culture was used to grow a 750mL large culture (1:500 dilution). The incubation at 37 degree was stopped when OD600 was around 0.8. The culture was then cooled on ice and 100uL of it was taken as the reference before IPTG induction. The protein expression was induced with 0.5mM IPTG at 18 degree for 16 hours with shaking. 100uL of culture was taken after the induction to assess the expression of the protein. The E.coli cells were harvested by centrifuging the culture at 8000rpm, 4 degree for 10min each time. Each pellet was resuspended in 12mL resuspension buffer (300mM NaCl + 20mM Tris (pH =8)). The E.coli cells were then lysed by adding ~5mg lysozyme and fast freezing in liquid nitrogen for 15min.
5uL DNAseI, 15uL 1M MgCl2 and 3uL 1M CaCl2 were added into thawed E.coli cell lysate to digest the DNA. The lysate was left in room-temperature water bath for 1 hour until it became less viscous. 5uL lysate was taken as the reference before centrifuging for separation. The soluble and insoluble fractions of the lysate was separated by centrifuging at 14500rpm, 4 degree for 1h, and 5uL supernatant was taken to assess the solubility of the protein.
Purification of the recombinant protein generally took use of the His-tag at the end. 1mL Ni-NTA agarose equilibrated with resuspension buffer (300mM NaCl + 20mM Tris (pH = 8)) was used. 3 6mL elutions containing wider imidazole gradient was performed (10mM, 100mM, 250mM). TGX stain-free gel was ran to check the expression, solubility, and Ni column purification. For hmHRP, the expression of the protein is not significant according to the gel, and it’s also hard to determine its solubility and presence in Ni column elutions (Figure 1). But from Western Blot of anti-His labelled with HRP, it can be observed that some protein even though not a lot is in Ni elution 2 (Figure 2). Thus, the Ni elution 2 was concentrated using 5000 MWCO ultracentrifuge from Satorius to around 300uL and loaded onto size exclusion column 650. Same buffer was used. 3 peaks were observed on the chromatography (Figure 3), and the fractions were loaded onto a SDS-PAGE gel with load to see their content. The gel was then blotted with anti-His labelled with HRP. The Blot shows that fraction 3 contains hmHRP (Figure 4). The chromatography calculates the concentration of fraction 3 to be only 1.57uM, so it was concentrated using 10000 MWCO ultracentrifuge from Satorius to around 300uL. The concentration was once again determined by Nanodrop to be 3.29uM. The fraction after concentration was used for later experiments.
Figure 3. Anti-His labelled with HRP of hmAvi gel filtration load and fraction 3, 5, 6, 7. The expected size of hmAvi is 21 kDa.
Figure 4. TGX stain-free gel of hmHRP. The expected size of hmHRP is 54 kDa
Electrophoretic gel mobility shift (EMSA)
MBD constructs were characterized by electrophoretic mobility shift assay (EMSA) (see Ref 10 for references). In this method, protein and DNA will be pre-incubated and run on a native polyacrylamide gel. If protein binds to DNA, DNA will be slowed while running; at the same time, a “retardation band” will form when the gel is visualized with DNA-specific dye. DNA not incubated with the proteins was run along the same gel as standards.
Figure 5. Retardation bands were present only symmetrically methylated DNA probes (lane 3), suggesting its specificity.
24 bp primers containing two methylation sites in close proximity were chosen as the target to test protein-DNA binding, in order to characterize specificity of different protein constructs. To be more clinically relevant, one of the methylation sites on the primer was cg10428836, which was shown to have significant methylation level change in hepatocellular carcinoma (HCC) (14). The other methylation site, by contrast, has little known clinal relevance. Forward and reverse primers were pre-hybridized to dsDNA before the assay; in the figures, primers were specially labeled as like “F 0_0”, which indicates that it was a forward primer with no methylation on both sites “0”. Another example will be “R M_0”, which stands for reverse primer with a site methylated near 5’ end “M” (this methylation site is cg10428836).
Pre-hybridization was performed for: no methylation (both 0_0), hemi-methylation (0_0 + M_0), symmetrical methylation (M_0, M_0), and unsymmetrically methylation (0_M + M_0). A good protein construct will only bind to symmetrically methylated DNA but not hemi-methylated nor unsymmetrically methylated ones; latter will be the noises for our device.
Hm-HRP protein demonstrated a good specificity toward symmetrically methylated DNA (Figure 1, protein: DNA molar ratio = 1.75:1). To confirm this result, Hm-HRP on the same gel was transferred to nitrocellulose membrane and subsequently probed with anti-His labeled HRP. The membrane was then visualized with Western Femto substrate (Figure 2 to show that Hm-HRP was present in the retardation band.
Figure 6. Hm-HRP was transferred and visualized with anti-His antibody. Hm-HRP/DNA complex was confirmed by the presence of protein signal at lane 3.
Complementary platform
MBD domain is good at detecting methylated CpG sites, therefore it is suitable for methylation detection; but two challenges are present when a sensitive and quantitative MBD assay is required, such as characterization of ctDNAs. First of all, quantitative methylation assay typically has an output of methylation percentage such as beta value, which requires knowledge of total DNA content. This remains unknown when the assay only captures MBD-DNA binding activity. Secondly, MBD2 binding is sequence-nonspecific, meaning that it could not differentiate between different methylation sites. This will become a crucial problem when multiple methylation sites are present in the DNA substrates, common for most methylated DNA. In our project, we introduced and characterized a graphene oxide (GO) platform to overcome these two challenges. Details of the platform could be referred to 2018 UC San Diego Wiki page and original reference of the platform were provided (12). In brief, we coupled MBD binding assay with a total DNA quantification fluorescent assay, utilizing GO’s fluorescent quenching property. Differentiation of specific sites are achieved by using complementary fluorescent ssDNA probes. We further improved our design by introducing exonuclease III signal amplification strategy. This platform aims to complement our fusion MBD parts to fully quantify methylation levels of specific CpG sites on ctDNA. We also anticipate MBD parts coupled with this platform will be useful for future projects to quantify other methylated DNA substrates.
References
1. Hendrich B, Bird A (November 1998). "Identification and Characterization of a Family of Mammalian Methyl-CpG Binding Proteins". Mol Cell Biol. 18 (11): 6538–47. PMC 109239. PMID 9774669
2. E. Li, T. H. Bestor, R. Jaenisch, Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69, 915-926 (1992).
3. P. W. Laird, R. Jaenisch, THE ROLE OF DNA METHYLATION IN CANCER GENETICS AND EPIGENETICS. Annual Review of Genetics 30, 441-464 (1996).
4. D. Macleod, R. R. Ali, A. Bird, An Alternative Promoter in the Mouse Major Histocompatibility Complex Class 11 I-A3 Gene: Implications for the Origin of CpG Islands. Molecular and Cellular Biology 18, 4433-4443 (1998).
5. Sun, Chang, Laura L. Reimers, and Robert D. Burk. “Methylation of HPV16 Genome CpG Sites Is Associated with Cervix Precancer and Cancer.” Gynecologic oncology 121.1 (2011): 59–63. PMC. Web. 18 Oct. 2018.
6. Epigenomics. 2015;7(6):1051-73. doi: 10.2217/epi.15.39. Epub 2015 Apr 30.
7. Guezennec, Xavier (2006). "MBD2/NuRD and MBD3/NuRD, Two Distinct Complexes with Different Biochemical and Functional Properties". Molecular and Cellular Biology. 26 (3): 843–851. doi:10.1128/MCB.26.3.843-851.2006. PMC 1347035. PMID 16428440. Retrieved 2018-09-05.
8. Y. Yu, S. Blair, D. Gillespie, R. Jensen, D. Myszka, A. H. Badran, I. Ghosh, A. Chagovetz, Direct DNA Methylation Profiling Using Methyl Binding Domain Proteins. Analytical Chemistry 82, 50125019 (2010).
9. M. F. Fraga, E. Ballestar, G. Montoya, P. Taysavang, P. A. Wade, M. Esteller, The affinity of different MBD proteins for a specific methylated locus depends on their intrinsic binding properties. Nucleic Acids Research 31, 1765-1774 (2003).
10. Heimer, Brandon Walter, Methylated DNA detection using a high-affinity methyl-CpG binding protein and photopolymerization-based amplification. http://hdl.handle.net/1721.1/98796
11. Robert J. Klose, Shireen A. Sarraf, Lars Schmiedeberg, Suzanne M. McDermott, Irina Stancheva, Adrian P. Bird, DNA Binding Selectivity of MeCP2 Due to a Requirement for A/T Sequences Adjacent to Methyl-CpG, Molecular Cell, Volume 19, Issue 5, 2005, Pages 667-678, ISSN 1097-2765, https://doi.org/10.1016/j.molcel.2005.07.021. (http://www.sciencedirect.com/science/article/pii/S1097276505015091)
12. Lu Chun-Hua, et al. A Graphene Platform for Sensing Biomolecules. Angewandte Chemie International Edition. Vol 48, Issue 26. Jun2009. https://doi.org/10.1002/anie.200901479
13. Shu-yan Niu, Quan-yi Li, Rui Ren, Shu-sheng Zhang, Enzyme-enhanced fluorescence detection of DNA on etched optical fibers, Biosensors and Bioelectronics, Volume 24, Issue 9, 2009, Pages 2943-2946, ISSN 0956-5663, https://doi.org/10.1016/j.bios.2009.02.022. (http://www.sciencedirect.com/science/article/pii/S0956566309001067)
14. Zhang K, et al. Circulating tumor DNA methylation markers for diagnosis and prognosis of hepatocellular carcinoma. Nature material. Oct 2017. DOI: 10.1038/NMAT4997.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 882
Illegal XhoI site found at 1444 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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