Difference between revisions of "Part:BBa K3378888"
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Leucine-rich ameloblast peptide (LRAP) is an alternative splicing product expressed by ameloblast genes during enamel development. LRAP has N-terminal amino acids and C-terminal amino acids of whole amelogenin, so it has the similar characteristics to the total length of amelogenin.  
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<p>Leucine-rich ameloblast peptide (LRAP) is an alternative splicing product expressed by ameloblast genes during enamel development. LRAP has N-terminal amino acids and C-terminal amino acids of whole amelogenin, so it has the similar characteristics to the total length of amelogenin. </p>
  
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===Usage and Biology===
 
===Usage and Biology===
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<p>LRAP can self-assemble into a chain structure composed of nanospheres by relying on its C-terminal domain, and the presence of calcium ions can enhance its ability. Non-phosphorylated LRAP tends to induce the formation of ordered hydroxyapatite (HA), while phosphorylated LRAP  tend to stabilize amorphous calcium phosphate (ACP) and prevent their conversion to HA in vitro. So the non-phosphorylation LRAP are sufficient to transform ACP into ordered bundles of apatite crystals.We also found that LRAP has the ability to bind to natural teeth.</p>
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
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===Functional Parameters===
 
===Functional Parameters===
 
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<p>To obtain the LRAP protein, we constructed pET-28a(+)-LRAP and transferred it to <i>E. coli </i>BL21(DE3) (<b>Figure 1</b>). The <i>E. coli</i> strain was cultured to OD<sub>600</sub>=0.6, induced with 0.5 mM IPTG, and allowed to grow overnight at 16 ℃. LRAP’s molecular weight is about 6.5kDA. We infered that LRAP can be expressed in <i>E. coli</i> at the condition described above by Tris-Tricine-SDS-PAGE analysis(<b>Figure 2</b>). </p>
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<img src=https://2020.igem.org/wiki/images/3/37/T--HZAU-China--LRAPF1.jpg width=100% alt="">
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<center><b>Figure 1.</b> Linear pET-28a(+) and LRAP obtained by PCR .</center>
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<div style="width: 45%; margin: 20px auto">
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<img src=https://2020.igem.org/wiki/images/c/c7/T--HZAU-China--LRAPF2.jpg width=100% alt="">
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<center><b>Figure 2.</b> Tris-Tricine-SDS-PAGE analysis of LRAP expression.</center>
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<p>Since LRAP protein was difficult to be purified, we cultured BL21(DE3) strain without plasmid <b>(WT)</b>, uninduced BL21(DE3) strain with plasmid<b>(-IPTG)</b> and induced BL21(DE3) strain with plasmid<b>(+IPTG)</b>.And then crushed the bacteria by ultrasonication, centrifuged at 10000xg and 4℃ for 10min, and took the supernatant for follow-up experiments. <br>
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To demonstrate the mineralization capacity of LRAP, the supernatant was added into artificial saliva(8.7mM KCl,0.6mM MgCl<sub>2</sub>,10.0mM CaCl<sub>2</sub>,4.6mM K<sub>2</sub>HPO<sub>4</sub>,2.7mM KH<sub>2</sub>PO<sub>4</sub>) to make the final protein concentration was 5mg/ml. The solution was incubated at 37℃ for 24h and the turbidity was observed(<b>Figure 3</b>).We observed that the +IPTG group had less precipitation and higher turbidity and we speculated the phenomenon is because that the formation of precipitation is prevented by the nucleation of LRAP and ions in artificial saliva.</p>
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<div style="width: 45%; margin: 20px auto">
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<img src=https://2020.igem.org/wiki/images/9/9d/T--HZAU-China--LRAPF3.jpg width=100% alt="">
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<center><b>Figure 3.</b>Effects of different supernatants on turbidity of artificial Saliva.</center>
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<p>We incubated the supernatant of ultrasound-broken bacteria which include LRAP together with pig teeth. Then we designed the experiment called “Toothtern-blot” to verify the existence and function of LRAP.<br>
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The teeth of domestic pigs were broken into fragments about 5mm long on each side, which  were selected and stored in 75% alcohol at room temperature.
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The total protein concentration of the three supernatants was adjusted into 10mg/mL. Three pieces of pig teeth were washed with PBS buffer to remove the surface’s alcohol, then put in three 2mL centrifuge tubes respectively. After that, three supernatants with uniform protein concentration were added respectively. Incubate at 37℃ for 20h, and the tubes were upside  down for several times every 5h to eliminate the influence of protein deposition processes as much as possible.<br>
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The incubated pig teeth fragments were repeatedly washed with PBS buffer for 2-3min to remove the protein that was not bound to the teeth.The rinsed pig teeth were incubated in plastic petri dishes with diameter of 10cm, 50 mL 5% skimmed milk was used to diluted His-tag, Rabbit pAb (YEASEN Biotech,Shanghai, China) at 1:10000 and hybridized with teeth at room temperature for 2h. Then  washed them with TBST buffer 3 times for 10min each. After that, 50 mL 5% skimmed milk was used to diluted  Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) (YEASEN Biotech,Shanghai, China) at 1:10000 and hybridized with teeth at room temperature for 2h. Then washed them with TBST buffer 3 times for 10min each. Use A Super ECL Detection Reagent ECL (YEASEN Biotech,Shanghai, China) for development operations.
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The results showed that LRAP was able to bind to the tooth fragments and had binded more in the dentin(<b>Figure 4</b>).</p>
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<br>
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<div style="width: 45%; margin: 20px auto">
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<img src=https://2020.igem.org/wiki/images/c/c4/T--HZAU-China--LRAPF4.jpg width=100% alt="">
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</div>
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<center><b>Figure 4.</b>"Toothtern-blot" analysis of different supernatants. <br> <b>A and B. </b>Pictures of same samples in different exposures.  <b>C. </b>Picture of the teeth in reality.</center>
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[1] Kwak S Y, Litman A, Margolis H C, et al. Biomimetic enamel regeneration mediated by leucine-rich amelogenin peptide[J]. Journal of dental research, 2017, 96(5): 524-530.<br>
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[2] Le Norcy E, Kwak S Y, Wiedemann-Bidlack F B, et al. Leucine-rich amelogenin peptides regulate mineralization in vitro[J]. Journal of dental research, 2011, 90(9): 1091-1097.
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Latest revision as of 02:29, 28 October 2020


LRAP(Leucine-rich Amelogenin Peptides)

Leucine-rich ameloblast peptide (LRAP) is an alternative splicing product expressed by ameloblast genes during enamel development. LRAP has N-terminal amino acids and C-terminal amino acids of whole amelogenin, so it has the similar characteristics to the total length of amelogenin.

Usage and Biology

LRAP can self-assemble into a chain structure composed of nanospheres by relying on its C-terminal domain, and the presence of calcium ions can enhance its ability. Non-phosphorylated LRAP tends to induce the formation of ordered hydroxyapatite (HA), while phosphorylated LRAP tend to stabilize amorphous calcium phosphate (ACP) and prevent their conversion to HA in vitro. So the non-phosphorylation LRAP are sufficient to transform ACP into ordered bundles of apatite crystals.We also found that LRAP has the ability to bind to natural teeth.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 132
  • 1000
    COMPATIBLE WITH RFC[1000]

Functional Parameters

To obtain the LRAP protein, we constructed pET-28a(+)-LRAP and transferred it to E. coli BL21(DE3) (Figure 1). The E. coli strain was cultured to OD600=0.6, induced with 0.5 mM IPTG, and allowed to grow overnight at 16 ℃. LRAP’s molecular weight is about 6.5kDA. We infered that LRAP can be expressed in E. coli at the condition described above by Tris-Tricine-SDS-PAGE analysis(Figure 2).

Figure 1. Linear pET-28a(+) and LRAP obtained by PCR .

Figure 2. Tris-Tricine-SDS-PAGE analysis of LRAP expression.

Since LRAP protein was difficult to be purified, we cultured BL21(DE3) strain without plasmid (WT), uninduced BL21(DE3) strain with plasmid(-IPTG) and induced BL21(DE3) strain with plasmid(+IPTG).And then crushed the bacteria by ultrasonication, centrifuged at 10000xg and 4℃ for 10min, and took the supernatant for follow-up experiments.
To demonstrate the mineralization capacity of LRAP, the supernatant was added into artificial saliva(8.7mM KCl,0.6mM MgCl2,10.0mM CaCl2,4.6mM K2HPO4,2.7mM KH2PO4) to make the final protein concentration was 5mg/ml. The solution was incubated at 37℃ for 24h and the turbidity was observed(Figure 3).We observed that the +IPTG group had less precipitation and higher turbidity and we speculated the phenomenon is because that the formation of precipitation is prevented by the nucleation of LRAP and ions in artificial saliva.

Figure 3.Effects of different supernatants on turbidity of artificial Saliva.

We incubated the supernatant of ultrasound-broken bacteria which include LRAP together with pig teeth. Then we designed the experiment called “Toothtern-blot” to verify the existence and function of LRAP.
The teeth of domestic pigs were broken into fragments about 5mm long on each side, which were selected and stored in 75% alcohol at room temperature. The total protein concentration of the three supernatants was adjusted into 10mg/mL. Three pieces of pig teeth were washed with PBS buffer to remove the surface’s alcohol, then put in three 2mL centrifuge tubes respectively. After that, three supernatants with uniform protein concentration were added respectively. Incubate at 37℃ for 20h, and the tubes were upside down for several times every 5h to eliminate the influence of protein deposition processes as much as possible.
The incubated pig teeth fragments were repeatedly washed with PBS buffer for 2-3min to remove the protein that was not bound to the teeth.The rinsed pig teeth were incubated in plastic petri dishes with diameter of 10cm, 50 mL 5% skimmed milk was used to diluted His-tag, Rabbit pAb (YEASEN Biotech,Shanghai, China) at 1:10000 and hybridized with teeth at room temperature for 2h. Then washed them with TBST buffer 3 times for 10min each. After that, 50 mL 5% skimmed milk was used to diluted Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) (YEASEN Biotech,Shanghai, China) at 1:10000 and hybridized with teeth at room temperature for 2h. Then washed them with TBST buffer 3 times for 10min each. Use A Super ECL Detection Reagent ECL (YEASEN Biotech,Shanghai, China) for development operations. The results showed that LRAP was able to bind to the tooth fragments and had binded more in the dentin(Figure 4).


Figure 4."Toothtern-blot" analysis of different supernatants.
A and B. Pictures of same samples in different exposures. C. Picture of the teeth in reality.



[1] Kwak S Y, Litman A, Margolis H C, et al. Biomimetic enamel regeneration mediated by leucine-rich amelogenin peptide[J]. Journal of dental research, 2017, 96(5): 524-530.
[2] Le Norcy E, Kwak S Y, Wiedemann-Bidlack F B, et al. Leucine-rich amelogenin peptides regulate mineralization in vitro[J]. Journal of dental research, 2011, 90(9): 1091-1097.