Abstract
Antigen Used for Immunization
To generate three different recombinant AtSIZ1 proteins, named F/D4R, D1F/D4R, and D1F/R, AtSIZ1 cDNA (locus no. At5g60410) were amplified with the following primers: F/D4R (5'-GAATTCATGGATTTGGAAGCTAATTGTAAGG-3' and 5'-GAATTCTTACTCAGAATCCGAGTCAATGGAG-3'), D1F/D4R (5'-GAATTCATGCAAGTATCTGGGGCAAGTG-3' and 5'-GAATTCTTACTCAGAATCCGA GTCAATGGAG-3'), and D1F/R (5'-GAATTCATGCAAGTATCTGGGGCAAGTG-3' and 5'-GAATTCAGATCTAAATAGTT GGAACCCAG-3'), respectively. A pair of primers was designed, with an EcoRI site at the 5'-end and 3'-end. Three amplified cDNA fragments were cloned into pET28a (Novagen, Darmstadt, Germany) and transformed into Escherichia coli strain BL21(DE3). The sequence was confirmed by DNA sequencing. The expression of recombinant protein and purification of different recombinant His-tagged AtSIZ1 proteins were performed according to the protocol provided by Novagen.
Three different recombinant AtSIZ1 proteins were expressed in E. coli, which were cultured in LB broth containing 100 mg/mL ampicillin. The cultures were incubated at 37°C with constant shaking, until the optical density at 600 nm reached 0.5–0.6. Recombinant AtSIZ1 protein was induced IPTG (0.6 mM) and further cultured at 37°C with constant shaking for 3 h. The bacteria were harvested by centrifugation at 9500 g for 20 min. The cell pellet was resuspended in 25 mL of buffer A (20 mM Tris-HCl [pH 8.0], 8 M urea) per gram of cells. The cells were disrupted by sonication and solublized at room temperature (RT) with constant shaking for 1 h. The soluble proteins were harvested by centrifuging at 9500 g and RT for 20 min. The supernatant was dialysed against buffer B (20 mM Tris-HCl [pH 8.0], filtered) using dialysis bags with a 6–8 kDa molecular weight cut-off. The dialyzed recombinant AtSIZ1 protein was centrifuged again at 4°C for 20 min in order to remove any aggregated proteins.
In the pET28a expression vector, the AtSIZ1 sequence was followed at its N-terminus by the six histidines for affinity purification using nickel chelate affinity chromatography. The supernatant was loaded onto a nickel-charged column (Clontech, Mountain View, CA), and the column was washed with 50 times bed volumes of buffer C (20 mM Tris-HCl [pH 8.0], 300 mM NaCl) containing 0.1% of TritonX-114 for endotoxin removal. Then, the His-tagged recombinant AtSIZ1 protein was eluted using buffer D (20 mM Tris-HCl [pH 8.0], 300 mM NaCl, 300 mM imidazole). The fractions containing the protein of interest were dialyzed against buffer B (20 mM Tris-HCl [pH 8.0], filtered) using the same dialysis bags. The protein was further purified by Superdex 75 HR 10/30 gel filtration (Amersham Pharmacia Biotech, Piscataway, NJ) using a FPLC system with buffer B (20 mM Tris-HCl [pH 8.0], filtered). The recombinant protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Method of Immunization
To generate monoclonal anti-AtSIZ1 antibodies, purified recombinant D1F/D4R protein was used to immunize female BALB/c mice (6 weeks of age) by subcutaneous injections. The mixtures of Freund's complete adjuvant (Sigma-Aldrich) and recombinant D1F/D4R protein was used for the two immunizations in 2 week intervals. The mixtures of Freund's incomplete adjuvant and D1F/D4R protein were used for the third immunization. Mice were bled 10 days after each boost. The titer of mouse serum was determined by direct enzyme-linked immunosorbent assay (ELISA). The last boost of antigen was injected 3 days before a fusion.
Parental Cell Line Used for Fusion
BALB/c myeloma cell line FO was obtained from ATCC and fused with splenocytes from immunized mice according to standard protocols.
Selection and Cloning Procedure
FO myeloma cell line was fused with splenocytes from immunized mice according to standard protocols. The hybridoma cells of positive wells were cloned by limiting dilution in aminopterin-free selection Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum. The single clones were screened by direct ELISA, dot blotting, and Western blotting for their specificity to recombinant AtSIZ1. A single hybridoma clone was expanded for further purification. Anti-AtSIZ1 MAb IgG was purified from hybridoma cell culture supernatants using protein G agarose beads (KPL, Gaithersburg, Maryland).
Heavy and Light Chains of Immunoglobulin
Not determined.
Specificity
By Western blot from recombinant D1F/D4R
The MAb clones 9, 10, 34, and 45 recognized two molecular sizes of recombinant D1F/D4R protein whereas clone 13 detected recombinant D1F/D4R as a single band but the detection signal was very weak. The MAb clone 24 detected only the lower band. The four MAb clones, 9, 10, 34, and 45 were able to react with full-length recombinant AtSIZ1 (F/DR4) protein and sensitivities were very similar. The MAb clone 9 detected recombinant AtSIZ1 (F/DR4) at 10 nanogram range.
By Western blot from endogenous AtSIZ1
The antibody detected endogenous AtSIZ1 in Arabidopsis wild-type and C-siz1-2 plant tissues with anti-AtSIZ1 MAb in a specific manner, but not in siz1-2 (deletion mutant) plant tissue. Thus, the specificity of anti-AtSIZ1 MAbs to endogenous AtSIZ1 was a reliable result with relatively low cross-reaction to other proteins. This result suggests that anti-AtSIZ1 MAb can recognize specifically endogenous AtSIZ1. However, the molecular size of a specific band is approximately 70 kDa, which is smaller than the molecular size of recombinant AtSIZ1 protein from full-length AtSIZ1 cDNA. This is presumably due to its post-translational modification. The number of protein isoforms in proteomes can be two to three orders of magnitude higher than the number of genes in the genomes. This is in large part due to post-translational modifications of proteins that provide covalent alterations to protein backbones and side chains that increase proteome complexities. Post-translational modifications modulate the activity of most eukaryote proteins. These modifications result in mass changes that are detected during analysis. Therefore, it is possible to regulate the AtSIZ1 activity as SUMO ligase through post-transcriptional protein modification.
Specific Antigen Identified
Not determined.