Difference between revisions of "Part:BBa K2924042"
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Small proteins are usually expressed in prokaryotic organisms like <i>Escherichia coli</i>, which enables easy, quick and cheap protein expression<sup>3</sup>. Disadvantages are the lack of post-translational modification and glycosylation, the difficult expression of large proteins and proteins with disulphide bonds<sup>3,4</sup>.
 
Small proteins are usually expressed in prokaryotic organisms like <i>Escherichia coli</i>, which enables easy, quick and cheap protein expression<sup>3</sup>. Disadvantages are the lack of post-translational modification and glycosylation, the difficult expression of large proteins and proteins with disulphide bonds<sup>3,4</sup>.
 
These drawbacks can be compensated by using eukaryotic yeast as an expression host. Two of the primarily used yeast expression systems are <i>Saccharomyces cerevisiae</i> and <i>Pichia pastoris</i> <sup>5</sup>.  
 
These drawbacks can be compensated by using eukaryotic yeast as an expression host. Two of the primarily used yeast expression systems are <i>Saccharomyces cerevisiae</i> and <i>Pichia pastoris</i> <sup>5</sup>.  
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===Usage and Biology===
 
  
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In <i>P. pastoris</i>, the AOX1 promoter is methanol inducible and therefore the AOX is highly expressed in the presence of methanol<sup>22</sup>. This leads to high recombinant protein yields of genes introduced into <i>P. pastoris</i> under influence of the AOX1 promoter<sup>23</sup>.  
 
In <i>P. pastoris</i>, the AOX1 promoter is methanol inducible and therefore the AOX is highly expressed in the presence of methanol<sup>22</sup>. This leads to high recombinant protein yields of genes introduced into <i>P. pastoris</i> under influence of the AOX1 promoter<sup>23</sup>.  
 
The pPICZB Vector from the EasySelect™ Pichia Expression Kit from Thermofisher Scientific was used, which contains an inducible AOX1 promoter and a Zeocin™ resistance gene.
 
The pPICZB Vector from the EasySelect™ Pichia Expression Kit from Thermofisher Scientific was used, which contains an inducible AOX1 promoter and a Zeocin™ resistance gene.
  
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The lactoferrin coding sequence originates from the organism <i>Bos Taurus</i> and was synthesized commercially. In addition, a NotI and an Eco72I interface were placed at both ends of the gene to clone the gene into the pPICZB vector (Fig. 36).
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The lactoferrin coding sequence originates from the organism <i>Bos Taurus</i> and was synthesized commercially. In addition, a NotI and an Eco72I interface were placed at both ends of the gene to clone the gene into the pPICZB vector (Fig. 1).
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[[File:Lactoferrin Pic1.png|500px|thumb|right|<i><b>Fig. 1:</b> The genetic organisation of the P. pastoris expression system with the AOX Promoter BBa_K2924039 followed by the target gene and C-terminally fused to a His-tag and AOX1 Transcription Terminator BBa_K2924040. The vector is pPICZB. E: Eco72I. N: NotI</i>]]
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===Usage and Biology===
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Revision as of 15:42, 20 October 2019


AOX promoter + Lactoferrin + HisTag + AOX Terminator

The heterologous expression of proteins has an important industrial role, e.g. for the production of insulin1. Identification of new peptide and protein pharmaceuticals and the optimization of the expression of known pharmaceuticals represent a huge research sector. Around 155 pharmaceuticals and vaccines developed by bio-pharmaceutical companies were approved by the US Food and Drug Administration in 2002, the amount of approved recombinant proteins quickly rose to over 200 in 20092. Besides insulin, medically important proteins like albumin and the human growth hormone (HGH) are produced by microbes or higher organisms. Small proteins are usually expressed in prokaryotic organisms like Escherichia coli, which enables easy, quick and cheap protein expression3. Disadvantages are the lack of post-translational modification and glycosylation, the difficult expression of large proteins and proteins with disulphide bonds3,4. These drawbacks can be compensated by using eukaryotic yeast as an expression host. Two of the primarily used yeast expression systems are Saccharomyces cerevisiae and Pichia pastoris 5.

In P. pastoris, the AOX1 promoter is methanol inducible and therefore the AOX is highly expressed in the presence of methanol22. This leads to high recombinant protein yields of genes introduced into P. pastoris under influence of the AOX1 promoter23. The pPICZB Vector from the EasySelect™ Pichia Expression Kit from Thermofisher Scientific was used, which contains an inducible AOX1 promoter and a Zeocin™ resistance gene.

The lactoferrin coding sequence originates from the organism Bos Taurus and was synthesized commercially. In addition, a NotI and an Eco72I interface were placed at both ends of the gene to clone the gene into the pPICZB vector (Fig. 1).

Fig. 1: The genetic organisation of the P. pastoris expression system with the AOX Promoter BBa_K2924039 followed by the target gene and C-terminally fused to a His-tag and AOX1 Transcription Terminator BBa_K2924040. The vector is pPICZB. E: Eco72I. N: NotI


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1
    Illegal BamHI site found at 2232
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1220
    Illegal BsaI.rc site found at 2215