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Revision as of 20:37, 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.

The structure of a haptoglobin-haemoglobin complex.

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.

The fractions obtained from IMAC were pooled and concentrated to 500 µl and loaded onto a Superdex 75 10/300 column to carry out size exclusion chromatography. Results from this can be seen in Figure 2.

Figure 2: Further characterization of human haptoglobin following SEC. A) Chromatogram showing the elution profile of the His-tagged human haptoglobin. The fractions corresponding to the two peaks observed on the chromatograph were further analysed by SDS page gel and western blotted. B) 10µl of the fractions A7-A8 and A8-A9 corresponding to peaks 1 + 2, respectively were mixed with 10µl of Laemmli buffer and loaded onto a 12.5% SDS-PAGE gel. Band A observed on the gel corresponds to the expected size of haptoglobin – 45kDa. However, it is not clear what the bands present at ~37kDa may be, possibly haptoglobin that has lost its His-tag. C) Samples separated by SDS-PAGE were transferred to a nitrocellulose membrane and probed with an anti-his antibody. This Western blot analysis confirmed the presence of human haptoglobin.

To further identify the expressed protein at ~45kDa, the band was sent for analysis by tryptic peptide mass spectrometry. This technique uses the proteolytic enzyme trypsin which cleaves at the carboxy side of arginine and lysine residues. The sizes of the peptide fragments obtained after trypsin digestion, represent the peptide mass fingerprint and are characteristic of each protein. The peptide mass fingerprint spectrum of fragments derived through trypsin digest indicated that haptoglobin was indeed purified with good coverage of the amino acid sequence to the expected sequence (Figure 3). This shows that we have purified two forms of our protein, one containing the histidine tag and the other which has lost the tag.

Figure 3: Mass spectrometry analysis of haptoglobin. The expected sequence of haptoglobin is shown here with the matched peptides from the mass spectrometry analysis shown in bold red.

Sequence and Features