| [OBJECTIVES]LIM domains are characterized by two cysteine-composed double-zinc finger motifs, which mediate protein-protein interactions. According to the presence of additional motifs, LIM proteins are grouped into the four main familis: nuclear-only, LIM-only, LIM-actin associated, and LIM-catalytic. In general, LIM proteins play important roles in a variety of biologic processes such as organ development, cytoskeleton reorganization, and cell differentiation. The "Four and a Half LIM domain (FHL) proteins" belong to the LIM-only protein family, comprising the five proteins FHL1, FHL2, FHL3, FHL4, and ACT. Human FHL2, originally known as DRAL (Down-regulated in Rhabdomyosarcoma LIM protein), was first identified by subtractive hybridization of normal myoblasts versus a rhabdomyosarcoma cell line. Under physiologic conditions, FHL2 exhibits a tissue-restricted expression pattern with prominent expression in heart, prostate epithelial cells and human ovary. FHL2 is able to interact with a broad spectrum of functionally proteins related to several different signaling pathways. It have been demonstrated that FHL2 is a coactivator of androgen receptor(AR), estrogen receptor (ER), cAMP response element-binding protein(CREB), cAMP-responsive element modulator(CREM). activator-protein-1 (AP-1), NF-κB, beta-catenin, hNP220, Focal adhesion kinase ,WT1 ,ERK2, hCDC47, Insulin-like Growth Factor-binding Protein 5 (IGFBP-5) and TNF receptor-associated factor 6 (TRAF6). Evidence was obtained that FHL2 was associated with tumor development and apoptosis. Recent studies reported that high levels of FHL2 protein in patients with lung, ovary, Breast Cancer, gastric and colon cancers were correlated positively with tumor metastasis and invasion. Taken together, available evidences suggest that FHL2is an important prognostic molecular marker in a variety of cancers. To further investigate the function and putative clinical applications of this protein, it is urgent to develop antibody with high specificity and sensitivity. In current study, we expressed FHL2 protein in E. coli, purified the recombinant protein, generated polyclonal antibody and evaluated its application in research areas.[METHODS]1. Construction of pET22b/FHL2Cellular total RNA was isolated from colon cancer cell line, Lovo cells. Reverse transcription-polymerase chain reaction (RT-PCR) was performed in accordance with the manufacturer' instructions. The primers used were sense 5'- AAC GAATTC C ATGACTGAGCGCTTTGACTG- 3' and 5'-TTT GTCGAC GATGTCTTTCCCACA GTCGG- 3'. The sense and antisense primers contained the cutting sites of EcoR I and Sal I restriction endonuclease, respectively. The PCR conditions were initial denaturation at 94℃for 5 min, followed by denaturation at 94℃for 30 sec, annealing at 55℃for 30 sec, and extension at 72℃for 45 sec for 6 cycles and denaturation at 94℃for 30 sec, annealing at 65℃for 30 sec, and extension at 72℃for 45 sec for 26 cycles, with the final extension at 72℃for 10 min after the last cycle of amplification. The PCR-generated DNA was restricted with EcoR I and Sal I and subcloned into prokaryotic expression vector pET22b+ (Novagen) at the same cutting sites. The reconstructed plasmid was tranformmed into E.coli, amplified and identfied by both double cleaved with EcoR I and Sal I and gene sequencing.2. Over-expression and Purification of Recombinant FHL2 ProteinThe pET/FHL2 plasmid was transformed into E. coli BL21 (DE3) (Novagen; Madison, WI) and plated on LB agar plates containing ampicillin (50μg/ml). After isopropyl-thio-β-D-galac-toside (IPTG) induction, the construct will proposed to express FHL2-His fusion protein that containing a 6XHis tag at the C-terminal of FHL2 protein. Ten colonies were selected by small-scale IPTG induction for the presence of a 37 KDa recombinant protein on Coomassie Blue-stained SDS-PAGE. The clone showing the strongest induction was selected for large-scale FHL2 protein purification. The strongest expressive colony was inoculated into LB liquid growth medium containing ampicillin (50μg/ml) and cultured at 37℃with shaking at 250 rpm. Over-expression of recombinant FHL2 was induced by supplementation of the growth medium with 1mM IPTG. After induction, the bacterial cells were harvested by centrifugation and re-suspended into 50ml UrNTA-0 Buffer (20mM Tris-HCl pH7.9, 0.5M NaCl, 10% Glycerol, 8M Urea). After sonication, cell debris was removed by centrifugation at 10,000rpm, 4℃for 15min and the supernatant was loaded onto a chelating sepharose column (15cm long, 1.6cm diam), charged with NiSO4 and equilibrated with UrNTA-0 Buffer. Bound proteins, due to the presence of the C-terminal poly-histidine tail, were washed with 250ml of UrNTA-0 Buffer. Recombinant FHL2 was eluted with a linear gradient of imidazole (UrNTA-0 containing imidazole from 20mM to 200mM). Purity was assessed by SDS-PAGE and fractions containing pure recombinant FHL2 (rFHL2) were dialysed by a 14 KDa filter with a linear decent of urea (PBS containing urea from 8M to 0M). The protein concentration was determined by the method of bicinchoninic acid (BCA) assay, using bovine serum albumin (BSA) as the standard.3. Immunizations and collection of IgGNew Zealand White rabbits were injected subcutaneously with 600μg (2ml) of purified rFHL2 mixed with an equal volume of complete Freund's adjuvant after preimmune serum had been collected. The immunization was repeated at 4 and 6 weeks after the first injection. The animals were bled 1 week after the third injection and then once monthly. Antibody titers were determined by ELISA, and the animals were sacrificed when there was no further incease in antibody titers. Serum was collected, IgG was separated and purified with ammonium sulfate precipitation. The purity of antibody (anti-FHL2) was assessed by SDS-PAGE.4. Detection of FHL2 protein with anti-FHL2 by Western blot Lovo cells were rinsed with PBS buffer, scraped off the plates, and pelleted by centrifugation. The cell pellets were resuspended in lysis buffer (20 mM Tris, pH 7.4, 140 mM NaCl, 0.1% Nonidet P-40, 1 mM PMSF, 1μg/ml each of pepstatin, leupeptin, and aprotinin) for 30 min. The total amounts of protein were determined by the BCA assay. A total of 20μg of protein for each sample was then mixed with 2×sample buffer, denatured at 95℃for 5 min, and separated on 12.5% SDS-PAGE. Proteins were then transferred to PVDF membranes (Milipore) followed by blocking with 1% BSA in PBS for 2 hr and then incubated for 1hr with anti-fhl2 IgG diluted at 1:125, 1:250, 1:500, 1:1000, 1:2000, at room temperature. The membranes were washed three times with PBS/T (PBS with 0.1% Tween-20) and then incubated for 1 hr with 1:2500 dilution of horseradish peroxidase-conjugated anti-rabbit antibody (Cell Signal). The antigen-antibody complexes were visulized by DAB (Boster) or the enhanced chemiluminescence (ECL) system from Amersham.5. Precipitatation of FHL2 protein with anti-FHL2 by ImmunoprecipitationColon cancer cell, SW480 was grown in six-well plates and transfected transiently with the PCI-3Xflag-FHL2 construct, which was kindly provided by the Institute of Molecular Biology, University of Hong Kong; and could express FHL2 protein with a 3Xflag tag in eukaryotic cells. 48 hr post-transfection, cell lysates were prepared. 800 g of cell lysate was incubated with 3 g of anti FHL2 antibody at 4℃for 3 hr for followed by incubation with protein A-agarose for additional 1 hr. The bead were washed, boiled at 95℃in SDS sample buffer for 5 min and blot by anti-flag.6. Staining of cellular FHL2 protein with anti-FHL2 by Immunofluorescence and ImmunocytochemistryGasric cancer cell, Kato-III were seeded onto tissue culture slides (Roth, Karlsruhe, Germany) to a density of 105 cells/cm2 and grown in RPMI 1640 over night. Fixed with 4% freshly prepared paraformaldehyde for 10 min on glass slides and then treated with 0.1% Nonidet P-40 for 5 min. Cells were pretreated with 5% normal goat serum for 60 min to block nonspecific bindings and then were incubated at room temperature for 60 min with anti-FHL2 IgG diluted to 1:50. Control samples were incubated with preimmune serum. After thorough rinses with PBS, for immunofluorescence slides were incubated with Texas Red-conjugated anti-rabbit secondary antibody for 45min (dilution 1:100). Nuclei were stained with Hoechst 22358(1μg/ml). Cells were analyzed using a fluorescent microscope. For immunocytochemistry slides were incubated with biotinylated anti-rabbit IgG secondary antibody followed by 3% peroxide block. After washing, the signals were developed using the avidin-biotin complex method/3,3'-diaminobenzidine detection kit (Boster) followed by hematoxylin counterstain.7. Immunohistochemistry of paraffin-embedded tissueStaining was done on 4-μm sections of paraffin-embedded tissue. Antigen retrieval was achieved by heating the slides in citrate buffer (pH 6.0; Biogenix, San Ramon, CA). Positive signals were obtained as using staining protocol similar to that immunocytochemistry mentioned above.[RESULTS]1,Construction of recombinant plasmid expressing FHL2Identical to the published FHL2 sequenced, 854bp of PCR product was obtained and successfully cutted by by EcoRI and SalI followed by the ligaiton to pET22b. Recombinant construct pET22b/FHL2 was generated. Gene sequencing Showed that the cloned sequence was completely identical to that of FHL2 published in Pubmed.2. Expression and Purification of Recombinant FHL2 ProteinpET22b/FHL2 was transformmed into E. coli, as shown in Fig.2 (A), induction of E. coli with IPTG resulted in a high level expression of a 37KDa protein corresponding to the molecular mass of rhFHL2. The protein was concentrated and refolded. The estimated recovery of purified rhFHL2 protein was 1.2mg/ml.3. Production of Polyclonal AntibodyPurified rhFHL2 protein was injected into three New Zealand White rabbits. The highest titre of anti-serum obtained was 1:125,000 in two rabbits. The concentration of purified anti-fhl2 IgG is 2mg/ml determined by BCA kit. Only one band appeared in SDS-PAGE in assessing the purity of anti-fhl2 IgG. 4. Detection of FHL2 Protein with Anti-fhl2 by Western BlotColon cancer cell lines Lovo and SW480, gastric cancer cell line Kato-III are of human origin. When the lysates of these cells were assayed for expression of FHL2 with anti-fhl2 by western blot, as shown in Fig.2, only one specific band appeared with the relative molecular weight of approximately 40 KDa. The highest dilution of anti-FHL2 for western blot analysis was 1:2,000.5. Immunoprecipitation with anti-FHL2 IgGAnti-FHL2 prepared was capable of precipitating target protein. As shown in Fig.4, a distinct protein that migrates with a relative molecular weight of 40 KDa was visulized when co-immunoprecipitation assay was applied into the lysates of SW480 cells..6. Immunofluorescence staining with anti-FHL2 IgGWe performed immunofluorescence analysis of SW480 cells. Immunofluorescence staining of the fixed SW480 cells with 1:50 diluted IgG anti-fhl2 gave a nuclear and cytoplasmic signals that was absent in samples stained with preimmune serum.7. Immunocytochemistry taining with anti-FHL2 IgGWhen stained with anti-FHL2 IgG , KatoIII cells gave a very strong nuclear signal and weak cytoplasmic signal. On the contrary, the control slides which stained with preimmune as primary antibody had no any stain in cells.8. Immunohistochemistry of Paraffin-embeded Tissue Samples with anti-FHL2 IgGTo evaluate the role of anti-FHL2 IgG in detection of FHL2 by immunohistochemistry, we choice a paraffin-embeded tissue specimen derived from a colon cancer xenografts in situ in nude mice. We showed that intense positive signal was observed after staining by immunohistochemistry assay.[CONCLUSIONS]1. Recombinant human FHL2 (rhFHL2) protein was successfully expressed in E.coli. 2. Pure with high titre of anti-FHL2 polyclonal antibody was prepare in immunized rabbits.3. The anti-FHL2 antibody was effectively and specifically in detection of FHL2 protein by western blot (minimal comcentration: 1:2000), immuniprecipitation, immunofluorescence/immunocytochemistry and immunohistochemistry.The antibody obtained was of specific and senstive. It will facilitate the future studies in assessing the function and clinical application of FHL2. |
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