Baculovirus carrying the PARP1 gene from zebrafish and recombinant drPARP1 (Danio rerio PARP1) protein were generated during my master thesis.
4.1.1 RNA Isolation from Fish
Medaka and killifish RNA was isolated from internal tissue as ovary or testis, liver, spleen, kidney and muscle, avoiding the gastro-intestinal tract. The RNA was isolated following the protocol of Chomczynski and colleagues (3.2.1), (Chomczynski and Sacchi, 1987). Each isolation process resulted in two to six RNA samples (Appendix Table 6-1). The concentration of the RNA was measured photometrically and the quality was determined by absorbance ratio of Abs260nm / Abs280nm and agarose gel electrophoresis (Figure 4-1).
Figure 4-1 Quality test of total fish RNA. 1 µg of total RNA from different fish species were separated using RNA agarose gel electrophoresis. 24 RNA samples were tested. White boxes indicate chosen RNAs. M = Marker (GeneRuler 1kb DNA ladder, LifeTechnologies)
The RNAs show contamination with genomic DNA in varying degrees: from very high (RNA: 1 – 5) to low (RNA: 10, 13, 15, 20, 28) to no contamination (RNA:
31, 32). The relation between the 28S rRNA and the 18S rRNA, which should be 2 : 1, yields information about the quality and the degree of degradation. All RNA samples, except RNA 9, 19, 20 and 27, display a stronger 28S rRNA band than 18S rRNA band. Considering these criteria one RNA sample of each fish species was chosen and used for further experiments. For olPARP1 (Oryzias latipes PARP1) cDNA production RNA no. 15 was taken. RNA no. 19 was used for further experiments regarding N.furzeri, RNA no. 10 for N.orthonotus, RNA no. 28 for N.rubripinnis and RNA no. 2 for N.korthausae.
4.1.2 FishPARP1 Sequence Information
In order to design primer for cDNA production and amplification it is essential to know the genomic or mRNA sequence. The PARP1 sequence of medaka and of zebrafish were retrieved from databases (www.ensembl.org and www.ncbi.nlm.nih.gov). cDNA sequences of the Nothobranchius species were obtained via RACE (rapid amplification of cDNA ends) during this work. In Figure 4-2 the schematic PARP1 mRNA with primers necessary for RACE, reverse transcription and PCR is depicted.
Figure 4-2 Schematic overview of PARP1 mRNA with different primers. The yellow arrow indicates the PARP1 cDNA with 5´- and 3´-UTR and poly(A)-tail. Purple arrows display primers, the arrowhead indicates forward or reverse orientation. Primers above the sequence were used to bind to PARP1 of Nothobranchius species, below the sequence to medaka PARP1. RACE primer: AAP = abridged anchor primer, UAP = universal amplification primer, AUAP = Abridged universal amplification primer, those binding independent of cDNA.
First a 3´-RACE was performed for all Nothobranchius species. After the reverse transcription with the gene-unspecific RACE primer (33), the specific PARP1 cDNA amplifications were done using the AUAPrimer (34) and the internal primer (8). This internal primer binds about 650 bp before the C-terminal end of the sequence. It was designed by comparing PARP1 sequences of multiple fish (zebrafish, medaka, fugu, salmon, stickleback and tetraodon) searching for areas with maximum identity. The PCR products were sent for sequencing. Using this information two more primers were designed in the 3´-UTR (untranslated region) near the end of the sequence: Primer (46) for reverse transcription and PCR of N.furzeri and primer (47) for N.orthonotus, N.korthausae and N.rubripinnis. Primer (14) was designed the same way as (8) and binds to the PARP1 coding sequence at position 151. Using sequence-specific primer (46) or (47) for reverse transcription and PCR plus primer (14) as sequence-specific forward primer it was possible to obtain further information of the coding sequence. This sequence information was used for designing oligonucleotides (36), (38) and (41), which were a necessary prerequisite for the subsequent 5´-RACE of all Nothobranchius species. On the basis of the sequence information of the 5´-UTR the sequence-specific primer (44) was designed and together with primer (46) / (47) it was possible to amplify the whole PARP1 base sequences of the different Nothobranchius species.
4.1.3 Bacterial Cloning Vector carrying fishPARP1 cDNA
In order to subclone the PARP1 cDNAs, they were introduced into the vector pSL1180. In a first step a reverse transcription was carried out using gene-specific primers (O.l.: (17), N.f.: (46), N.k., N.o. and N.r.: (47)). PCR was carried out for all fish species using phosphorylated forward and reverse primers located outside of the coding sequence. For medaka oligonucleotides (32) and (17) were used, for N.furzeri (44) and (46) and for the other killifish (44) and (47). After amplification and separation via agarose gel electrophoresis the PARP1 cDNA was retrieved from the agarose gel (3.2.7). The cloning vector pSL1180 was digested using the blunt cutting restriction enzyme EcoRV and
dephosphorylated to avoid religation. Vector and inserts were ligated and chemo – competent E.coli DH5α were transformed with the newly built plasmid.
To distinguish between clones carrying a plasmid containing the PARP1 sequence (3.6 kbp, Figure 4-3, lane 1, 4-6, 9, 10) and those without (0.45 kbp, Figure 4-3, lane 7 and 8) colony screen PCR was performed (3.2.3). Positive clones were grown in liquid LB medium and after plasmid purification the plasmids were sequenced stepwise to confirm the correct sequence and orientation, which was reverse in all cases.
Figure 4-3 Representative colony screen PCR of pSL1180-PARP1 (N.orthonotus). Primer (50) and (51) were used, templates were bacteria picks. The PCR products were run on a 0.8 % agarose gel. M = Marker (GeneRuler 1kb DNA ladder, LifeTechnologies)
4.1.4 Baculoviral Vector carrying fishPARP1 cDNA
In the next step the fishPARP1-pSL1180 plasmids were used to subclone the PARP1 cDNA into baculoviral expression vectors in order to use the baculovirus expression system for overexpression of recombinant PARP1 protein from the fish species (Figure 4-4).
Therefore, the vectors pVL1392 (medaka) and pBacPAK8 (Nothobranchius species) were used. Baculoviral vectors were opened by using the restriction endonucleases XmaI and XbaI and PARP1 cDNAs were isolated from pSL1180 plasmids via the same enzymes. Of note, pSL1180 carrying inserts was
additionally digested by ApaLI to ensure fragmentation of the plasmid backbone. After transformation, insert integration was tested by colony PCR using the primer-pair (52) / (53).
Figure 4-4 Schematic subcloning strategy. FishPARP1 was cut out by restriction digest (XmaI, XbaI, ApaLI) and cloned into opened (XmaI, XbaI) vector backbone of pBacPAK8 or pVL1392. Restriction sites are indicated using scissors. Working steps are stated in the boxes.
The PARP1 coding sequence is highlighted in yellow.
Plasmid DNA from positive clones was purified and used for generating baculoviral clones containing the respective PARP1 cDNA (3.5.1). Single virus clones were isolated via Plaque Assay and then amplified twice in Sf9 cells to achieve the necessary virus concentration in the supernatant (~9 x 106 pfu / ml) (3.5.2 & 3.5.4).
4.1.5 Overexpression and Purification of recombinant fishPARP1
PARP1 overexpression of all fish species was carried out in 2x 10 cm dishes each. The purification procedure was optimized for minimal time requirement, i.e. 36 h from harvesting of infected Sf9 cells to storage of purified recombinant protein (3.6.3). The purification process had to be monitored for proper
down-stream applications, i.e. by determining suitable fractions containing PARP1 from the Sephadex-column. The samples were analyzed (Figure 4-5, A) and PARP1-positive samples (typically fractions 4 – 8) were used in the further purification process. These fractions were applied on a DNA cellulose column for affinity purification (Figure 4-5, B).
Figure 4-5 Purification of recombinant PARP1. Representative western blots of a PARP1 purification batch (N.furzeri). (A) 1.5 ml fractions 1 – 14 after Sephadex column (size separation). (B) Samples after DNA cellulose column (separation by affinity). 4 % - 20 % SDS-PAGE (BioRad) followed by H250C-WB; a = before dialysis, b = after dialysis, FT = flow through, S = supernatant after first purification step (cell lysis).
After dialysis of the elution fraction the PARP1 concentration was determined using the Whitaker and Granum method (3.6.4). The following table (Table 4-1) gives an overview of recombinant PARP1 protein concentrations ranging from 28 ng / µl (N.korthausae) to 82 ng / µl (zebrafish):