Difference between revisions of "Part:BBa K3889022"
 
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<partinfo>BBa_K3889022 short</partinfo>
 
<partinfo>BBa_K3889022 short</partinfo>
  
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Since ovastacin (<partinfo>BBa K3889024</partinfo>) has a eukaryotic origin, this protein undergoes several Post Translational Modifications (PTMs) including the addition of disulphide bonds and phosphorylation at serine and tyrosine residues.  
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Since Ovastacin (<partinfo>BBa_K3889024</partinfo>) has a eukaryotic origin, this protein undergoes several Post Translational Modifications (PTMs) including the addition of disulphide bonds and phosphorylation at serine and tyrosine residues.  
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However, our recombinant protein will be produced in a prokaryotic organism which lacks the necessary genes to carry out PTMs. Hence, we used phosphomimetic variant(s) of ovastacin by changing or mutating the serine to aspartic acid and tyrosine to glutamic acid residues at S99, S156, S244 and Y279 (amino acid numbered with reference to pro-ovastacin) respectively. We then used I-TASSER to predict the structure of this phosphomimetic variant.  
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However, the recombinant protein if produced in a prokaryotic organism will lack the necessary machinery to carry out PTMs. Hence, a phosphomimetic variant(s) of Ovastacin was made by changing or mutating the serine to aspartic acid S99, S156 and S244 (amino acid numbered with reference to pro-Ovastacin).
  
 
===Protein Sequence===
 
===Protein Sequence===
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RLLSAASNKWPMGGDGVVEVPFLLSSKYDEPSRQVILEALAEFERSTCIRFVTYQDQRDFISIIPMYGCFSDVGRSGGMQVVSLAPTCLQKGRGIVL<strong>HELMHVLGFWH</strong>E HTRADRDRYIRVNWNEILPGFEINFIKSRSSNMLTPYDYSSVMHYGRLAFDRRGLPTITPLWAPSVHIGQRWNLSASDITRVLKLEGCS
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<html>
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<head>
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<style>
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div.scroll {overflow-x: aut0; overflow-y: hidden;}
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</style>
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</head>
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<body>
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<div class="scroll">
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<p>RLLSAASNKWPMGGDGVVEVPFLLSSKYDEPSRQVILEALAEFERSTCIRFVTYQDQRDFISIIPMYGCFSDVGRSGGMQVVSLAPTCLQKGRGIVL<span style="color:red">HELMHVLGFWH</span>EHTRADRDRYIRVNWNEILPGFEINFIKSRSSNMLTPYDYSSVMHYGRLAFDRRGLPTITPLWAPSVHIGQRWNLSASDITRVLKLYGCS</p>
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</div>
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<p>where <span style="color:red">red</span> denotes the active site [1]</p>
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<div class="scroll">
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<img src="https://static.igem.org/mediawiki/parts/8/8e/T--IISER-Tirupati_India--ovastacin_allignment.jpg">
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</div>
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</body>
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</html>
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Fig source: Geneious version 2021.2 created by Biomatters. Available from https://www.geneious.com
  
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[[File:T--IISER-Tirupati India--Fig3-Phosphomimic Ovastacin.png]]<br>
 
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Fig 3. Phosphomimetic variant of Ovastacin (aspartic acid residues marked with lime and glutamic acid residue marked with cyan)
 
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{| class=wikitable
 
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!C-score
 
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! Estimated TM-score
 
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! Estimated RMSD
 
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|-
 
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| 1.84
 
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| 0.97±0.05
 
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| 1.8±1.5Å
 
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|}
 
  
  
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<partinfo>BBa_K3889022 parameters</partinfo>
 
<partinfo>BBa_K3889022 parameters</partinfo>
 
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===References===
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1. Anna D. Burkart, Bo Xiong, Boris Baibakov, Maria Jiménez-Movilla, Jurrien Dean; Ovastacin, a cortical granule protease, cleaves ZP2 in the zona pellucida to prevent polyspermy. J Cell Biol 2 April 2012; 197 (1): 37–44. doi: https://doi.org/10.1083/jcb.201112094

Latest revision as of 11:23, 19 October 2021


Human Ovastacin protease phosphomimic_A

Since Ovastacin (BBa_K3889024) has a eukaryotic origin, this protein undergoes several Post Translational Modifications (PTMs) including the addition of disulphide bonds and phosphorylation at serine and tyrosine residues. However, the recombinant protein if produced in a prokaryotic organism will lack the necessary machinery to carry out PTMs. Hence, a phosphomimetic variant(s) of Ovastacin was made by changing or mutating the serine to aspartic acid S99, S156 and S244 (amino acid numbered with reference to pro-Ovastacin).

Protein Sequence

RLLSAASNKWPMGGDGVVEVPFLLSSKYDEPSRQVILEALAEFERSTCIRFVTYQDQRDFISIIPMYGCFSDVGRSGGMQVVSLAPTCLQKGRGIVLHELMHVLGFWHEHTRADRDRYIRVNWNEILPGFEINFIKSRSSNMLTPYDYSSVMHYGRLAFDRRGLPTITPLWAPSVHIGQRWNLSASDITRVLKLYGCS

where red denotes the active site [1]

Fig source: Geneious version 2021.2 created by Biomatters. Available from https://www.geneious.com


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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



References

1. Anna D. Burkart, Bo Xiong, Boris Baibakov, Maria Jiménez-Movilla, Jurrien Dean; Ovastacin, a cortical granule protease, cleaves ZP2 in the zona pellucida to prevent polyspermy. J Cell Biol 2 April 2012; 197 (1): 37–44. doi: https://doi.org/10.1083/jcb.201112094