Fig 1: Secretion of PTH peptides: supernatants. (A) Tricine SDS-PAGE of supernatants from cultures of control and experimental strains. Aliquots of 10 µl of each supernatant were loaded. (1): 1 µl of PTH84 (1 µg); (2): BL21(DE3) (pExAB; pET-PTH84); (3): BL21(DE3) (pET-PTH84); (4): protein ladder (5 µl); (5): BL21(DE3) (pExAB); (6): BL21(DE3) (pExAB; pET-PTH34); (7): BL21(DE3) (pET-PTH34); (8): 1 µl of PTH34 (1 µg). Asterisks, bands analysed by mass spectrometry. (B) Mass spectrometry analyses from bands marked in A. PTH84-6xHis and PTH34-6xHis sequences were included in the data base of the analysis. Red, peptides detected by mass spectrometry
Fig 2: Sequences containing the gene fusions cloned into pET-26b(+). Nucleotide sequences of the inserts and of the vector adjacencies are indicated. In grey and boxed, restriction sites employed for cloning the fusions. Start and stop codons are in bold and Shine-Dalgarno sequences are underlined. The amino acid sequence of the fusion products is indicated. Sequences corresponding to SDV and PTH variants are in blue and red, respectively. (A ) sdv-pth34-6xhis (229 bp). (B) sdv-pth84-6xhis (374 bp)
Fig 3: Intracellular PTH peptides: cell lysates. (A) Tricine SDS-PAGE of BL21(DE3) (pExAB; pET-PTH84) lysate. Lines: 1, 1 µl of PTH84 (1 µg); 2, 10 µl of cell lysate. (1) and (2), bands analysed by mass spectrometry. (B) Mass spectrometry analyses. Data base for band (1) included the unprocessed SDV-PTH84-6xHis sequence, and for band (2), the processed PTH84-6xHis sequence. Red, peptides detected by mass spectrometry. Arrow, specific cleavage site performed by the MccV T1SS
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