Difference between revisions of "Part:BBa K1590001"

Latest revision as of 11:33, 19 September 2015

Human Haemoglobin B

Human haemoglobin (hemoglobin) forms a tetramer consisting of two A-chains and two B-chains. This biobrick is a synthetic gene encoding the human haemoglobin B-chain that has been optimized for expression in E. coli.

Usage and Biology

Haemoglobin is the tetrameric protein molecule in red blood cells that carries oxygen. It is composed of four polypeptide chains, which in adults consist of two alpha (A) globin chains and two beta (B) globin chains. The protein also normally contains the iron-containing cofactor haem (or heme). Haemoglobin can still be found free in the blood plasma at a concentration of up to 0.1 g/l.

Results

Overexpression and Purification of Haemoglobin Beta

Initial experiments were carried out to optimise expression of haemoglobin beta (hHBB) within our E.coli chassis. The synthetic gene was subcloned into the pQE80-L overexpression vector that adds an N-terminal hexa-histidine tag onto the N-terminus of the protein. We found the following to be the optimum conditions:

We subcultured 50 µl of an overnight culture into fresh LB containing the appropriate antibiotics and grew the cells at 37C until an OD-600 of ~0.6 was reached. The production of protein was then induced by adding 1 mM (final concentration)of IPTG then the cells were grown for a further 3 hours at 37C. Next, 1 ml of this culture was then taken for analysis by western immunblot which showed that haemoglobin beta was successfully overexpressed (Figure 1).

Figure 1: Western analysis of Human Haemoglobin B production in E. coli.

Purification of haemoglobin beta

This was then scaled up and 4 litres of E. coli containing pQE80-L hHBB was grown for purification of haemoglobin B by immobilized metal affinity chromatography (IMAC). The optimum conditions for this were as follows: 4 x 1L of fresh LB growth medium containing ampicillin and kanamycin was inoculated with 50ml of haemoglobin beta overnight culture and left to grow until an OD₆₀₀ of between 0.6-1 at 37°C. The expression of protein was then induced by adding 1mM of IPTG then the cells were grown for a further 6 hours at 20°C. Cells were then pelleted and washed in a buffer of 50mM Tris-HCl pH7.5. Cells were then lysed and centrifuged to remove any cell debris. A crude extract was loaded onto a nickel affinity column and the protein was eluted with an imidazole gradient The results from this can be seen in Figure 2.

Figure 2: Purification of haemoglobin beta by immobilized metal affinity chromatography (IMAC). Our sample was loaded onto a nickel affinity column and the protein was eluted with an imidazole gradient.

The fractions corresponding to the IMAC peak were retained and concentrated down to 500 μl for further purification by size exclusion chromatography (SEC) - Figure 3.

Figure 3: Characterisation of haemoglobin B following SEC (size exclusion chromatography). A) The sample of concentrated fractions containing haemoglobin beta from nickel affinity purification was loaded onto an SEC column and the protein was eluted. B) 10µl of each fraction corresponding to the two observed peaks was mixed with 10µl of laemmli buffer and loaded onto an SDS gel. The bands observable on the gel are in line with the expected size of haemoglobin beta - 16kDa. C) Western Blotting was then carried out against an anti-his antibody to confirm the presence of haemoglobin B.

We have succesfully purified haemoglobin B and our next steps were to express and purify its binding partner haptoglobin.

Sequence and Features