Protein ladder on western blot dnak4/17/2024 ![]() Numbers to the left represent protein markers (Fermentas) in kDa.Ĭo-expression of PfAdoMetDC with plasmodial Hsp70s and supplementary GroEL/ES improves quality of product. Lower panels: DnaK was detected using α-DnaK antibody, PfHsp70 was detected using α-PfHsp70 antibody and PfAdoMetDC (60 kDa) was detected using α-Strep antibody. Lanes U–uninduced whole cell lysate I–whole cell lysate 1 hour post IPTG (1 mM) induction A1 –whole cell lysate 1 hour post AHT (2 ng/ml) induction A2 –whole cell lysate 2 hours post AHT plus IPTG induction. Whole cell lysates from the co-expression of PfAdoMetDC with chaperone sets (A) DnaK + DnaJ, (B) KPf + DnaJ, (C) PfHsp70 + DnaJ, (D) DnaK + GroEL + DnaJ, (E) KPf + GroEL + DnaJ, and (F) PfHsp70 + GroEL + DnaJ were analysed on SDS-PAGE (upper panel) and confirmed by Western blot analysis (lower panels). Numbers on the left handside represent protein markers (Fermentas) in kDa.Ĭo-expression of PfAdoMetDC with various chaperone sets in E. In each case, the left and right hand side lanes represents the sample that was taken before induction and 5 hours after induction with 1 mM IPTG, respectively. The labels on the top of the gel panels represent the different proteins that were expressed as well as the vector control (pQE60). Cells transformed with plasmid vector (pQE60) were used as negative control. (B) SDS-PAGE and Western analyses for the exogenous expression of KPf and the respective substrate binding cavity derivatives in E. The negative control consisted of cells transformed with pQE60 plasmid vector whilst the positive control was represented by cells transformed with the pQE60/DnaK plasmid. coli dnaK756 cells transformed with plasmid constructs expressing KPf and its substrate binding cavity mutants were incubated at 37☌ and 43.5☌, respectively. The hydrophobic pocket mutation abrogates the function of KPf. coli co-chaperones as well as recognise substrates of malarial origin. This model proposes KPf as an ideal Hsp70 co-expression partner for production of malarial proteins in a bacterial host as it is likely to cooperate with E. coli co-chaperones, or malarial protein recognition by the respective Hsp70 protein, and three stars represents a strong likelihood. A single star represents a weak likelihood of cooperation with E. KPf is comprised of the ATPase domain of DnaK and the PfHsp70 substrate binding domain (SBD), connected by the linker. coli DnaK, PfHsp70 and their chimeric derivative, KPf. Schematic representation of the domain organisation of E. ![]() coli co-chaperones and capability to recognise recombinant malarial proteins. ![]()
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