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===Functional Parameters===
 
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To further demonstrate that Azurin generated from our engineered bacteria could combine with p53 successfully, we mixed Azurin(without 6x His tag) with p53(with 6x His tag) and purified them together by a Ni-NTA column. The SDS-PAGE gel of combination verification shows below in <b>Figure 16</b>. Two correct stripes can be observed in the lane of the gel, which illustrates these two proteins can combine with each other.
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To demonstrate that our engineered bacteria could systhesis Azurin successfully, we designed this composite part and transformed the plasmid into <i>E.coli</i> BL21. The phoA label is utilized to assist Azurin being secreted into the medium, and His tag could combine with a Ni-NTA column. We purified Azurin by a Ni-NTA column and the SDS-PAGE gel is shown below in <b>Figure 1</b>. Correct stripe can be observed in the lane of the gel, which illustrates we obtained Azurin successfully.
 
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<center><img src="https://static.igem.org/mediawiki/parts/c/c0/T--HZAU-China-Azurin-comp-1.jpeg" style="width:300px;height:500px"></center>
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<center><b>Figure 1.</b> The SDS-PAGE gel of Azurin.</center>
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===Sequence and Features===
 
===Sequence and Features===

Latest revision as of 18:57, 21 October 2021

PhoA-azurin with a His-tag

Azurin is a copper-containing redox protein secreted by Pseudomonas aeruginosa. Azurin preferentially enters a variety of cancer cells and induces apoptosis or cell cycle arrest, thereby playing an anti-cancer effect. A 6×His tag was added in order to purify the protein. PhoA, a signal peptide of Sec pathway in Escherichia coli, have the capability to guide the LL-37 excrete out of the cell.

Functional Parameters

To demonstrate that our engineered bacteria could systhesis Azurin successfully, we designed this composite part and transformed the plasmid into E.coli BL21. The phoA label is utilized to assist Azurin being secreted into the medium, and His tag could combine with a Ni-NTA column. We purified Azurin by a Ni-NTA column and the SDS-PAGE gel is shown below in Figure 1. Correct stripe can be observed in the lane of the gel, which illustrates we obtained Azurin successfully.

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Figure 1. The SDS-PAGE gel of Azurin.

Sequence and Features

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]


Reference

Huang F, Qianhui S, Zhaojie Q, et al. Anticancer actions of azurin and its derived peptide p28[J]. The protein journal, 2020, 39(2): 182-189.