In this master’s thesis, soluble HbD-2-turboGFP fusion protein was expressed in E. coli Origami 2(DE3). Fluorescence
measurements of the fusion protein indicated a correctly folded and active turboGFP fusion-partner.
Fluorescence measurements also showed a much higher protein concentration in bacteria grown under IPTG
induction, than in the uninduced reference. The fusion protein size was confirmed on an SDS-PAGE gel.
The pET-11a-DEFB4A-GFP vector was constructed by ligation of DEFB4A-GFP and pET-11a, however, normal
subcloning strategies were not performed, as the DEFB4A-GFP gene had an on-gene NdeI restriction site.
Thus, an NdeI partial digestion of DEFB4A-GFP was first carried out, but this was unsuccessful.
Then a PCR site-directed point mutation was performed to mutate the on-gene NdeI restriction sequence from
CATATG to CATTTG. This was successful, and the mutated gene could be digested with NdeI and BamHI
restriction enzymes and ligated into digested pET-11a to form pET-11a-mut-DEFB4A-GFP vector.
Sequencing data of the constructed vector showed no mutations other than the point-mutation produced.