Difference between revisions of "Part:BBa K1590002"

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<b>Purification of haptoglobin</b>  
 
<b>Purification of haptoglobin</b>  
  
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The aim of iGEM Dundee 2015 was to design a cell-free system using highly pure hatoglobin. This requires overexpression of the synthetic gene and purification, via an engineered affinity tag, of the recombinant protein. Human haptoglobin was overproduced and purified by immobilized metal affinity chromatography (IMAC). The chromatograph obtained (not shown) showed two peaks: Peak 1 (fractions A2-A4) and Peak 2 (fractions A8-A11), these were then run on an SDS gel shown below in figure 1.  
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The aim of iGEM Dundee 2015 was to design a cell-free system using highly pure hatoglobin. This requires overexpression of the synthetic gene and purification, via an engineered affinity tag, of the recombinant protein. Human haptoglobin was overproduced and purified by immobilized metal affinity chromatography (IMAC). Eluted fractions were analysed by SDS-PAGE and Western immunoblotting (Figure 1).  
  
 
[[image:Dundee15_Haptoglobin2.jpg|thumb|left|500px|<b>Figure 1: Purification of human haptoglobin by nickel IMAC.</b>  A crude extract was loaded onto a nickel affinity column and the protein was eluted with an imidazole gradient (0-1000 mM). The fractions corresponding to a peak were collected. <b>A)</b> 10 µl of each fraction was mixed with 10 µl of Laemmli buffer and loaded onto an SDS gel. The bold bands observable in fractions A9-A11 are in line with the expected size of haptoglobin - ~45kDa.  <b>B)</b> Samples separated by SDS-PAGE were transferred to a nitrocellulose membrane and probed with an anti-His antibody to confirm the presence of haptoglobin.]]
 
[[image:Dundee15_Haptoglobin2.jpg|thumb|left|500px|<b>Figure 1: Purification of human haptoglobin by nickel IMAC.</b>  A crude extract was loaded onto a nickel affinity column and the protein was eluted with an imidazole gradient (0-1000 mM). The fractions corresponding to a peak were collected. <b>A)</b> 10 µl of each fraction was mixed with 10 µl of Laemmli buffer and loaded onto an SDS gel. The bold bands observable in fractions A9-A11 are in line with the expected size of haptoglobin - ~45kDa.  <b>B)</b> Samples separated by SDS-PAGE were transferred to a nitrocellulose membrane and probed with an anti-His antibody to confirm the presence of haptoglobin.]]
  
  
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<span class='h3bb'><b>Sequence and Features</b></span>
 
<span class='h3bb'><b>Sequence and Features</b></span>
 
<partinfo>BBa_K1590002 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1590002 SequenceAndFeatures</partinfo>

Revision as of 19:37, 18 September 2015

Human Haptoglobin

Haptoglobin is a human protein with high affinity for haemoglobin. This biobrick is a synthetic gene optimized for expression in E. coli.

Usage and Biology

In normal human blood plasma, haptoglobin circulates and binds to any free haemoglobin released from red blood cells. This is very important in normal physiology since free haemoglobin has potential damaging oxidative activity. The tight haptoglobin-haemoglobin complex can then be removed by the reticuloendothelial system, which is a part of the immune system. Engineered haptoglobin therefore has the potential to bind to, and potentially allow detection of, any free haemoglobin found in the environment.

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iGEM Dundee 2015

This synthetic gene was found to produce stable product when expressed in E. coli cells.

Results

Overexpression and purification of Human Haptoglobin

Purification of haptoglobin

The aim of iGEM Dundee 2015 was to design a cell-free system using highly pure hatoglobin. This requires overexpression of the synthetic gene and purification, via an engineered affinity tag, of the recombinant protein. Human haptoglobin was overproduced and purified by immobilized metal affinity chromatography (IMAC). Eluted fractions were analysed by SDS-PAGE and Western immunoblotting (Figure 1).

Figure 1: Purification of human haptoglobin by nickel IMAC. A crude extract was loaded onto a nickel affinity column and the protein was eluted with an imidazole gradient (0-1000 mM). The fractions corresponding to a peak were collected. A) 10 µl of each fraction was mixed with 10 µl of Laemmli buffer and loaded onto an SDS gel. The bold bands observable in fractions A9-A11 are in line with the expected size of haptoglobin - ~45kDa. B) Samples separated by SDS-PAGE were transferred to a nitrocellulose membrane and probed with an anti-His antibody to confirm the presence of haptoglobin.



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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1114
  • 1000
    COMPATIBLE WITH RFC[1000]