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Fig. 2 | Microbial Cell Factories

Fig. 2

From: Using a marine microalga as a chassis for polyethylene terephthalate (PET) degradation

Fig. 2

Secretion analysis of PETase-FLAG. a Schematic of the expressed recombinant protein AP_SP-PETaseR280A-FLAG. b Western Blot after SDS-gel separation of the cell pellet (10 µg of total protein) and medium fractions (total precipitated protein fraction) of 50 ml cultures (induced at OD600 = 0.4) expressing AP_SP-PETaseR280A-FLAG. Detection of recombinant proteins was conducted using an antibody against the FLAG-tag (α-FLAG). As control for intracellular proteins, an alpha-tubulin antibody (α-Tubulin) was used. Wild type medium and cell pellet fractions as well as a FLAG positive control lysate (Rockland, FLAG+) served as control protein fractions. AP_SP-PETaseR280A-FLAG clone 2 showed the highest expression and secretion efficiency (middle), whereas complete secretion of the recombinant protein was only achieved by clone 1 (left). As shown by the control via alpha-tubulin detection (right), presence of PETaseR280A-FLAG in the medium fraction was not due to cell lysis. A signal in the range of the calculated molecular mass of AP_SP-PETaseR280A-FLAG (30.4 kDa) could only be observed for clone 2 (left and middle). The dominant signals detected by the FLAG-tag antibody appeared at molecular masses of approximately 40 and 50–55 kDa in AP_SP-PETaseR280A-FLAG clone 1, 2 and 3. Calculated molecular masses: AP_SP-PETaseR280A-FLAG: 30.4 kDa; FLAG-tag, 1 kDa; PETaseR280A-FLAG: 28.5 kDa; FLAG+, 60 kDa. AP alkaline phosphatase, SP signal peptide, WT wild type, AP_# AP_SP-PETaseR280A-GFP clone #. Numbers beside/on the Western Blots indicate molecular masses of the marker (PageRuler™ Prestained 10–180 kDa Protein Ladder) bands in kDa

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