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Supplemental materials 2 – Cloning, expression and purification of recombinant
TOP1 fragments
1. Materials and Methods
1-1. RT-PCR
Total RNA was extracted from MCF-7 cells by using TRIzol reagents in
accordance with the manufacturer’s instructions (Life Technologies, Rockville, MD,
USA).
The quality of the total RNA was assessed by using formaldehyde gel
electrophoresis.
Next, 0.5 µg of the total RNA was reverse transcribed into cDNA
using a Superscript II Reverse Transcriptase kit (Fermentas Life Sciences, Schwerte,
Germany) in accordance with the manufacturer’s instructions.
Subsequently, cDNA
for amino acid sequence 1-765 of Homo sapiens DNA TOP1 (NCBI accession:
NP_003277.1) was randomly divided into 4 fragments by PCR with primers
containing BamH/HindⅢ restriction sites on both ends.
The primer sequences,
performing parameters, amplified nucleic acid sequences, and amino acid sizes are
listed in Table 1.
1-2. Expression of TOP1 fragments
We directionally cloned the PCR products into PQE 30 UA expression vectors
(Qiagen, Germany), and examined their DNA sequences before transformation of the
cloned plasmids into compete E coli M15 cells (Qiagen).
We cultivated the cells at
37°C in Luria Bertani (LB) media containing kanamycin (25 μg/mL) and ampicillin
(100 μg/mL), and induced the expression vectors by the addition of 1 mM of IPTG
for 3 hours at 37°C after the culture density reached A 600 nm = 0.6.
1-3. SDS-PAGE analysis
After induction of the expression vectors, we centrifuged the cells followed by
sonicated decellularization. We analyzed the supernatant of the sonification with
SDS-PAGE, which was stained with Coomassie brilliant blue and compared to
non-induced samples.
1-4. Purification of recombination TOP1 fragments
After induction, we harvested the cells by centrifugation, and resuspended the
pellet in Tris/HCl buffer (pH 8.0).
Next, we broken down the nucleic acids by mild
sonification and purified the recombinant proteins with a Ni2+-nitrilotriacetic acid
(Ni-NTA) resin (Qiagen).
We analyzed the expression of the recombinant proteins
by SDS-PAGE followed by immunoblots using anti-SCL-70 autoantibodies and the
rabbit antibody against the novel TAA, respectively.
2. Results
SDS-PAGE analysis showed that the plasmids expressed corresponding sizes of
recombinant proteins (SFig. 2a). SFig. 2b illustrates the amino acid sequences of the
4 recombinant fragments deduced from DNA sequencing after cloning into the
vectors.
3. Discussion and Conclusion
By successfully cloning and expressing 4 protein fragments of human TOP1, we
were able to determine whether the novel TAA was part of TOP1 and whether
anti-SCL-70 autoantibodies were different from the autoantibodies against the novel
TAA in cancer patients.
Table 1. Primer sequences and amplification conditions of PCR for cloning TOP1 fragments
TOP1 Fragments
Primers
PCR conditions
Amplified
sequences
1
2
3
4
F: 5’-CCGGATCCATGAGTGGGGACCAC-‘3
30 cycle: 94℃ ,30s, 56℃, 247-873 bp
R: 5’-AGGGGTACCCTCTTCTTCCCACCAT-‘3
30s, 72℃ ,1 min 30s
F: 5’-ATGGATCCAAAAAGAAGCCGAAG-‘3
30 cycle: 94℃ ,30s, 56℃, 820-1491 bp
R: 5’-CTCGGTACCGGAAACCAGCCAAGT-‘3
30s, 72℃ , 1 min 30s
F: 5’-ATGGATCCGTTACTTGGCTGGTT-‘3
30 cycle: 94℃ ,30s, 56℃, 1474-1920 bp
R: 5’-CTCGGTACCCTTGTTCTCCATAAA-‘3
30s, 72℃ ,1 min 20s
F: 5’-ACGGATCCCTATTTATGGAGAACAA-‘3
30 cycle: 94℃ ,30s, 56℃, 1921-2544 bp
R: 5’-CGCGGTACCCTAAAACTCATAGTC-‘3
30s, 72℃ ,1 min30s
The grey highlighted sequences in the primers were designed for subcloning into BamH/HindⅢ sites of the vector PQE30 UA.
RNA Corresponding
Protein sizes
209 aa
224 aa
149 aa
208 aa
5. Legends
SFig. 2a SDS-PAGE analysis of 4 recombinant fragments of TOP1.
Lanes 2, 4, 6
and 8 are IPTG induced samples of the fragments 1-4, respectively (as indicated by),
while lanes 1, 3, 5 and 7 are corresponding non-induced samples.
Molecular mass
markers (kDa) are shown on left side.
SFig. 2b The amino acid sequences of the 4 recombinant fragments of TOP1.
The
grey highlighted sequences are overlaps between the fragments when cloned by
RT-PCR.