Figure 3.

SDS-PAGE analysis and western blot analysis of protein (G-CSF) distribution between soluble and insoluble cell fraction after bacterial cell disruption. Target protein (G-CSF) was overexpressed in the form of ncIBs inside bacterial cells. Three mechanisms of bacterial cell disruption were studied: enzymatic lysis (L), high pressure homogenization (H) and sonication (S). After bacterial cell disruption soluble cell fraction (sn) and insoluble cell fraction (p) were separated with centrifugation and both samples were analysed with SDS-PAGE (A). Whole bacterial cells were also analysed (total cell proteins - Tcp). The most abundant protein in the samples lysed with enzymatic lysis is enzyme Lysozyme. Western blot analysis was also prepared to confirm the presence of target protein in individual fractions. Here two different analyses were prepared. The presence of protein in each fraction was confirmed with the western blot, where protein samples were reduced prior the analysis (B). Protein G-CSF is distributed between soluble and insoluble cell fraction when cells were disrupted with homogenization or sonication, whereas after enzymatic lysis almost all G-CSF is found in the insoluble cell fraction. The second western blot analysis was preformed with the protein samples without the addition of the reducing agent (C). Here the aggregation/polymerization of the protein G-CSF can be observed. Higher percentage of the aggregates can be observed in the insoluble sample (IBs) after sonication. Tcp - total cell proteins. Hsn - soluble cell fraction after homogenization. Hp - insoluble cell fraction after homogenization - Ibs. Ssn - soluble cell fraction after sonication. Sp - insoluble cell fraction after sonication - Ibs. Lsn - soluble cell fraction after enzymatic lysis. Lp - insoluble cell fraction after enzymatic lysis - Ibs.