Difference between revisions of "Part:BBa K3895007"
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==Construction==
 
==Construction==
 
Keratinase KerAVDZ50 (<partinfo>K3895003</partinfo>) is an extracellular serine thiol alkaline protease from the ''Actinomadura viridilutea'' strain, which can be isolated from the Algerian fishing port [1]. As a thermo-alkaline keratinases, it can function at a pH range of 7–12 and a temperature range of 35–80 °C. 6x His-tags were added to both sides of the sequence for purification and constructed into PET-28a(+). The constitutive T7 promoter (<partinfo>I719005</partinfo>) derived from the T7 bacteriophage allows high expression of proteins.
 
Keratinase KerAVDZ50 (<partinfo>K3895003</partinfo>) is an extracellular serine thiol alkaline protease from the ''Actinomadura viridilutea'' strain, which can be isolated from the Algerian fishing port [1]. As a thermo-alkaline keratinases, it can function at a pH range of 7–12 and a temperature range of 35–80 °C. 6x His-tags were added to both sides of the sequence for purification and constructed into PET-28a(+). The constitutive T7 promoter (<partinfo>I719005</partinfo>) derived from the T7 bacteriophage allows high expression of proteins.
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[[File:T--SZ_SHD--verify.png|900px|center]]
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==Protein purification==
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{|border=1 width="90%" align="center"
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|-
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!width="20%" style="background:#CCCCFF"|Name of keratinase
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!width="20%"|IPTG concentration/mM
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!width="20%"|Temperature/&deg;C
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!width="20%"|Time/h
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|-align="center"
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|style="background:#EEEEFF"|KerAVDZ50
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|0.67139
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|16
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|12
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|}
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[[File:T--SZ SHD--Z50plasmid.jpg|400px|center]]
 
[[File:T--SZ SHD--Z50plasmid.jpg|400px|center]]
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Revision as of 07:48, 15 October 2021


kerAvDZ50_dna_pET-28a(+)

Construction

Keratinase KerAVDZ50 (BBa_K3895003) is an extracellular serine thiol alkaline protease from the Actinomadura viridilutea strain, which can be isolated from the Algerian fishing port [1]. As a thermo-alkaline keratinases, it can function at a pH range of 7–12 and a temperature range of 35–80 °C. 6x His-tags were added to both sides of the sequence for purification and constructed into PET-28a(+). The constitutive T7 promoter (BBa_I719005) derived from the T7 bacteriophage allows high expression of proteins.

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Protein purification

Name of keratinase IPTG concentration/mM Temperature/°C Time/h
KerAVDZ50 0.67139 16 12
T--SZ SHD--Z50plasmid.jpg



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 606
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 411
    Illegal AgeI site found at 1149
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 351