Adult human hemoglobin (HbA) cloning and expression

It is generally known that the HbA is a tetrameric protein consisting of two subunits each of alpha- and beta-chain. Its main role is being an oxygen transporter. Regarding this, we investigated the HbA mutations, especially on the beta-chain, using a machine learning-based model and expressed generated mutants afterwards in the wet lab.

Initially, we started the laboratory part by assembling the ordered mutants (beta-chain gBlock fragments) into the DNA plasmid (pET-Duet) provided to us by our supervisor, Dr. Nélida Leiva Eriksson, consisting of the wild-type alpha and beta genes. The beta-chain mutants were substituted into the pET-Duet replacing the beta-chain of the wild-type gene. The substitution was simply done by restriction enzyme cloning, with NdeI and XhoI.

The assembled constructs were confirmed through colony PCR, PCR amplification of purified plasmid using a T7 primer and plasmid digestion using BamHI. Finally, four HbA mutants (labeled as D73A, D73K, K65D, and E6D) were confirmed to be successfully assembled in the pET-Duet.

The expression strain chosen was BL21-DE3. The expression was accomplished in 2 L baffled flasks with 500 mL Terrific Broth (TB) media by induction with 0.1 mM IPTG and 0.3 mM ALA the the time when the OD₆₂₀ reached ≥2.0. The induced cultivations were incubated at 28oC for 14 hours.

The identification of HbA protein

We were planning to determine the oxygen affinity of the HbA mutants by sending the purified HbA protein to Essex University. However, due to time limitations, only HbA identification and determining functionality were accomplished.

First, the cells expressing the mutants were sonicated and the crude supernatant was collected for SDS-PAGE analysis. An important step that needed to be considered during the cell disruption of HbA protein was the CO induction in between the sonication cycles. This is due to the fact that HbA is a labile protein and would otherwise not be detected in the analysis. The result from the SDS-PAGE is shown by fig. 1.

The size of the alpha and beta-chain are around 15 and 16 kDa, respectively. Thus, fig. 1 proves that the HbA mutants were expressed. Further identification as well as functionality analysis were conducted using a spectrophotometer, measuring a wavelength range from 350 to 650 nm. However, the plan was to purify the crude supernatant. Hence, an overnight dialysis as the initial step of purification was conducted prior to column chromatography. Due to the intensity of the red color of the crude supernatant being much less than that can be observed by the naked eye, 1 mL of each sample was diluted two times and analysed using spectrophotometry before continuing the purification. The results are shown by fig. 6-8 of this page.

The diluted sample then was placed in the cuvette, the absorbance was measured and considered as the oxygenated state and gave the maximum absorbance at 420, 550, and 570 nm. Into the same cuvette, sodium dithionite was added into the same cuvette in order to observe the reduced state of the HbA. The curve also shows that the peak of each sample was shifted from 420 to around 430 nm. This proves that the HbA was reduced. Further investigation was accomplished by bubbling CO into the cuvette for around 5 seconds to oxidize the reduced HbA. It was expected that the 430 nm peak shifted to the left. However, it was difficult to conclude about the oxidized state of the HbA. It could be due to the very low concentration of the HbA protein and impure sample. However, this analysis shows that the mutants are active.

Results and Discussion