Figure 1.
Top right insert - the exon and intron organization of PP13 DNA (LGALS13 or galectin 13) showing the 5 and 3 prime ends. The 4 exons are colored black (exon 1), blue (exon 2), orange (exon 3) and brown (exon 4). Figure body - the amino acid sequence of the anticipated protein with amino acid colored according to the respective exons. Red colored – the methionine added to the his tag. The sequences of the wild type recombinant PP13 (rPP13) (top line in each couple sequences) is constructed of 164 amino acids that are aligned with the sequence of the DelT221 (Truncated) (the lower of each couple), made of 98 amino acids. First line couple - the his-tag added to the N-terminal followed by the sequences of the first short exon (black), second exon (blue) and a part of the third exon (orange), which are identical in both molecules. Second line couple - continuation of exon 3 (orange) and beginning of exon 4 (brown) for the wild type and for the truncated variant lacking 28 amino acids of exon 3 and 7 amino acids of exon 4. Third line – the rest of exon 4 of the wild type, which is completely missing in the truncated variant (31 amino acids) and the C terminal tail, which is identical in both molecules.
Figure 2.
Purification and detection of the wild type and the truncated PP13.
A - Purification and characterization of heterologously expressed His-tagged truncated PP13. Expression of 6 His-tagged PP13 in E. coli was induced by IPTG. The protein was purified on Ni–NTA–agarose under denaturing conditions and separated on 15% SDS–PAGE. The total proteins were visualized by Gel Code blue staining. CL-clear lysate, Pel-cell pellet, Sup-Supernatant, FT-Flow through, W1 - 6 M urea, W2–4 M urea, W3 - without urea. B - The combined eluted fractions were separated by SDS-PAGE and stained by Gel-Code blue and the wildtype and truncated protein are shown. C - Proteins were electrotransferred by Western-blot. Only the His-tagged wild type (lane-1) but not the truncated PP13 (lane-2) was detected with the ELISA capture mAb (mAb1) and the detecting mAb (mAb2) as revealed with marking by HRP-conjugated rabbit anti mouse IgG and ECL detection reagent.
Figure 3.
A – Both 1st and 2nd mAbs recognize the wild type PP13 over background at 10 ng/ml (top panel). Using the same conditions the 1st mAb barely recognizes the truncated PP13 and the 2nd mAb doe’s not show a signal at all. B – Serial concentration of the wild type and truncated PP13 variants were placed on nitrocellulose, reacted with rabbit polyclonal antibodies to PP13 (pAbs) (top panel), or non-immune serum (NRS, middle panel) or without antibodies (HRP negative control) and reacted with goat anti rabbit IgG conjugated to HRP.
Figure 4.
Western blots with polyclonal pAbs.
The recombinant (rPP13) wild type PP13, its truncated variant (DelT221) and the native molecule (nPP13) purified from the placenta were separated by 15% SDS PAGE under non-reducing (A) or reducing (B) conditions, electro transferred to nitrocellulose and reacted with rabbit pAbs to PP13 (J67), pre-immune rabbit IgG (Jo) or without antibody (HRP negative control) followed by incubation with goat anti rabbit IgG conjugated with HRP. A single band is shown for the native and truncated PP13 in both reduced and non-reduced conditions. The recombinant wild type generates a single band in reduced conditions but in non-reduced conditions dimer, trimer and tetramer PP13 are revealed. The recombinant PP13 is heavier than the native protein due to the his-tag and extra tail sequence in the recombinant protein.
Figure 5.
Plates were coated with the wild type PP13 or the truncated variants at 2.5 µg/ml, blocked with 1% BSA in PBS and then incubated with serial dilutions of pAbs (A) or of the 1st, 2nd and anti Histidine mAbs followed by development with goat ant rabbit (A) or anti mouse (B) IgG conjugated to HRP. 5A – The signal is gradually reduced over a log dilution of the antibody concentration with either the wild type (diamonds, purple) or truncated (circles, blue) PP13 variant compared to the pre-immune response (diamonds and circles, respective colors). 5B – Both first (top panel) and second mAbs (middle panel) recognized the wild type PP13 (blue) but not the truncated (red) protein. Antibodies to the His-Tag (bottom panel) recognized both of the proteins.
Figure 6.
A. Mean arterial pressure (MAP) of pregnant rats after sub cutaneous implantation of slow releasing osmotic pumps with PP13 (black bars) (n = 9), the DelT221 mutant PP13 (dark gray bars) (n = 6) or saline (light gray bars) (n = 6). Mean values ± SD of MAP are presented from day 8 (before pump implantation), and after implantation on days 10, 13 and 15 of gestation (2, 5 and 7 days after pump implantation). P values show significances between saline controls and PP13 and saline controls and DelT221. B. The main uterine vein diameters of pregnant rats after pumps implantation is shown for 4 rats in each of the PP13 group (black bars), the DelT221 mutant group (dark gray bars) or saline control group (light gray bars). Mean values ± SD of the main uterine veins are presented for the 21st day of gestation (e.g. –9 days after the pumps were emptied from their releasing material). P values show significances compared to the saline control group and the DelT221 mutant group.