Difference between revisions of "Part:BBa K1660000"
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− | + | <p>A serine protease bace16 was first reported as a pathogenic factor against nematodes, whose accession number is AY708655. It was identified by methods such as ammonium sulfate precipitation. [1] In vitro assay demonstrated that the recombinant protease Bace16 expressed in Escherichia coli presented a nematotoxic activity, and it has been verified by experiments that Bace16 has the ability to degrade a nematode cuticle, leading to the nematode’s death.[2] Bace16 could be considered as a core component to kill the nematode.</p> | |
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===Usage and Biology=== | ===Usage and Biology=== | ||
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
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+ | <h2>Structure</h2> | ||
+ | The molecular mass of a mature Bace16 protein is about 28kDa. And the protein has 275 residues, with a catalytic triad center containing His, Asp, and Ser residues and two calcium binding sites for stabilizing the three-dimensional structure. Characterization of the purified protease revealed the optimum activity of Bace16 is at pH10, 50℃. The deduced protein also contains a presequence signal peptide of 30 amino acids and a propeptide of 77 amino acids. The presequence signal peptide directs the secretion of subtilisin from the interior of cells, while the propeptide functions as a chaperon to facilitate the folding process of the active protease.[3] | ||
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+ | <img width="60%" style="margin-left:180px" src="https://static.igem.org/mediawiki/2014/c/c6/Bnu_moda3.png"> | ||
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+ | <p class="fig">Figure 1 Expression and purification of ModA. Expression of ModA and its purification by His-chelating chromatography. Lane 1, molecular weight standards (kDa); lane 2, total bacterial proteins before IPTG induction; lane 3, total bacterial proteins after 0.5 mM IPTG induction; lane 4, total bacterial lysate after 0.5 mM IPTG induction; lane 5,the sediment after 16,000 rpm 30 min; lane 6 the flow through of column loading; lane 7,the flow of buffer B .lane 8 The purification of ModA by size-elution chromatography.</p> | ||
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+ | <img width="45%" style="margin-left:250px" src="https://static.igem.org/mediawiki/2014/e/e5/Bnu_moda4_01.jpg"> | ||
+ | <p class="fig" style="width:85%; margin-left:50px">Figure 2 Native-PAGE to provide the molybdate lane 1, the modA(acetic buffer PH 5.0); lane 2, the modA incubate with 20mM molybdate.</p> | ||
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Revision as of 11:40, 17 September 2015
Introduction
A serine protease bace16 was first reported as a pathogenic factor against nematodes, whose accession number is AY708655. It was identified by methods such as ammonium sulfate precipitation. [1] In vitro assay demonstrated that the recombinant protease Bace16 expressed in Escherichia coli presented a nematotoxic activity, and it has been verified by experiments that Bace16 has the ability to degrade a nematode cuticle, leading to the nematode’s death.[2] Bace16 could be considered as a core component to kill the nematode.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 125
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 65
Illegal XhoI site found at 151 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 247
Illegal NgoMIV site found at 610
Illegal NgoMIV site found at 682
Illegal NgoMIV site found at 934
Illegal AgeI site found at 541 - 1000COMPATIBLE WITH RFC[1000]
Structure
The molecular mass of a mature Bace16 protein is about 28kDa. And the protein has 275 residues, with a catalytic triad center containing His, Asp, and Ser residues and two calcium binding sites for stabilizing the three-dimensional structure. Characterization of the purified protease revealed the optimum activity of Bace16 is at pH10, 50℃. The deduced protein also contains a presequence signal peptide of 30 amino acids and a propeptide of 77 amino acids. The presequence signal peptide directs the secretion of subtilisin from the interior of cells, while the propeptide functions as a chaperon to facilitate the folding process of the active protease.[3]Figure 1 Expression and purification of ModA. Expression of ModA and its purification by His-chelating chromatography. Lane 1, molecular weight standards (kDa); lane 2, total bacterial proteins before IPTG induction; lane 3, total bacterial proteins after 0.5 mM IPTG induction; lane 4, total bacterial lysate after 0.5 mM IPTG induction; lane 5,the sediment after 16,000 rpm 30 min; lane 6 the flow through of column loading; lane 7,the flow of buffer B .lane 8 The purification of ModA by size-elution chromatography.
Figure 2 Native-PAGE to provide the molybdate lane 1, the modA(acetic buffer PH 5.0); lane 2, the modA incubate with 20mM molybdate.