Journal: Oncogene

Article Title: TAF1 acetyltransferase promotes colorectal carcinoma metastasis by catalyzing β-hydroxybutyrylation of KCTD9

doi: 10.1038/s41388-025-03644-1

Figure Lengend Snippet: A Tubacin decreases KCTD9 protein and increases KCTD9 K123bhb and K129bhb levels in HEK-293T and different CRC cell lines. HEK293T, HCT116, and LOVO cells were treated with Tubacin for the indicated times. Cell lysates were immunoprecipitated with anti-KCTD9 and analyzed by WB. B Tubacin has a negligible effect on KCTD9 mRNA levels. HEK293T, HCT116, and LOVO cells were treated with Tubacin for the indicated times. The relative KCTD9 mRNA levels were determined by qPCR. C , D Immunoblot and qPCR analysis of the KCTD9 K123bhb and K129bhb ( C ), and KCTD9 mRNA levels ( D ) in control and TAF1-knockdown HCT116 and LOVO cells. E Co-IP of endogenous TRIM21 with anti KCTD9 antibody in HCT116 cells. F Effects of TAF1 WT or HAT activity-deficient mutant Δ844-850 on KCTD9 association with TRIM21. Myc-TRIM21 and indicated Flag-TAF1 were co-transfected into HEK-293T cells with HA-KCTD9 constructs or empty vector control (EV). G Flag-tagged TRIM21 protein ubiquitinated HA-tagged KCTD9 analyzed by in vitro ubiquitination assays using purified recombinant proteins. Purified recombinant TRIM21 and TRIM21 ΔRING protein were incubated with ATP, E1, E2 proteins, ubiquitin, KCTD9-WT peptide and KCTD9-Kbhb peptide (10 μM or 20 μM) along with purified recombinant HA-tagged KCTD9 protein. H , I Effects of ectopic expression of KCTD9 WT or KCTD9 2KR mutant on KCTD9 protein degradation in HCT116 cells co-transfection of Myc-TRIM21 and Flag-TAF1 in exposure to cycloheximide (CHX, 100 μM) treatment (0, 30, 60, and 90 min). J Effects of KCTD9 WT, 2KQ or 2KR mutant on KCTD9 ubiquitination in HCT116/sgKCTD9 cells co-transfection of Myc-TRIM21 or/and Flag-TAF1. Data are representative of two or three independent experiments with similar results. Error bars, ± SD. * P < 0.05 or ** P < 0.01, by paired two-way Student’s t-test. n.s., negative significant.

Article Snippet: Antibodies for TAF1 (#12781), HES1 (#11988), CXCR4 (#64837), CXCL12 (#3740), activated Notch1 (NICD, #4380), CBP (#7389 T), P300 (#86377), TRIM21 (#92043), HA (#3724), Actin (#4967S) and His (#12698) were from CST (Danvers, MA); anti-β-Hydroxybutyryllysine (#PTM-1201RM) was from PTM BIO (Chicago, USA); antibody for KCTD9 (ab180937) were from Abcam Biotechnology (Abcam, Cambridge, UK); HEY1 (#19929-1-AP), MYC (#10828-1-AP), SNAI1 (#13099-1-AP) were from Proteintech; and anti-KCTD9 K123bhb and K129bhb rabbit polyclonal antibody generated by Proteintech using the peptide DKGVWGN-K(bhb)-QDHRGAF and D-K(bhb)-GVWGNK; and KCTD9-Kbhb peptide was synthesized by using the peptide of DK(bhb)GVWGN-K(bhb)-QDHRGAF; an antibody for FLAG (F3165) was from Sigma-Aldrich; antibodies for GAPDH (sc-25778) and GST (sc-138) were from Santa Cruz Biotechnology (Santa Cruz, CA); the secondary antibodies, anti-rabbit IgG, HRP-linked antibody (#7074) and anti-mouse IgG, HRP-linked antibody (#7076) were purchased from CST (Danvers, MA).

Techniques: Immunoprecipitation, Western Blot, Control, Knockdown, Co-Immunoprecipitation Assay, Activity Assay, Mutagenesis, Transfection, Construct, Plasmid Preparation, In Vitro, Ubiquitin Proteomics, Purification, Recombinant, Incubation, Expressing, Cotransfection

Journal: Oncogene

Article Title: TAF1 acetyltransferase promotes colorectal carcinoma metastasis by catalyzing β-hydroxybutyrylation of KCTD9

doi: 10.1038/s41388-025-03644-1

Figure Lengend Snippet: A Representative images of TAF1, KCTD9 K123bhb, KCTD9 K129bhb and NICD expressions in 113 clinical CRC primary tumor tissues. Scale bars, 50 μm. B Correlation of expression levels between TAF1, KCTD9 K123bhb, KCTD9 K129bhb and NICD in ( A ). C Prognosis comparison of CRC patients with TAF1/KCTD9-K123bhb, TAF1/KCTD9-K129bhb and TAF1/NICD ectopic differential expression using Kaplan-Meier survival analysis. D A working model of TAF1 promoted CRC metastasis through the KCTD9 Kbhb modification status. TAF1 directly binds to and catalyzes KCTD9 Kbhb modification at lys123 and lys129, thereby enhancing KCTD9 association with the E3 ubiquitin ligase TRIM21. This interaction leads to the promotion of KCTD9 degradation via the ubiquitin-proteasome pathway. KCTD9, as a tumor suppressor, its low levels activated Notch signaling pathway, ultimately contributes to the CRCSC properties, the progression of CRC and liver metastasis.

Article Snippet: Antibodies for TAF1 (#12781), HES1 (#11988), CXCR4 (#64837), CXCL12 (#3740), activated Notch1 (NICD, #4380), CBP (#7389 T), P300 (#86377), TRIM21 (#92043), HA (#3724), Actin (#4967S) and His (#12698) were from CST (Danvers, MA); anti-β-Hydroxybutyryllysine (#PTM-1201RM) was from PTM BIO (Chicago, USA); antibody for KCTD9 (ab180937) were from Abcam Biotechnology (Abcam, Cambridge, UK); HEY1 (#19929-1-AP), MYC (#10828-1-AP), SNAI1 (#13099-1-AP) were from Proteintech; and anti-KCTD9 K123bhb and K129bhb rabbit polyclonal antibody generated by Proteintech using the peptide DKGVWGN-K(bhb)-QDHRGAF and D-K(bhb)-GVWGNK; and KCTD9-Kbhb peptide was synthesized by using the peptide of DK(bhb)GVWGN-K(bhb)-QDHRGAF; an antibody for FLAG (F3165) was from Sigma-Aldrich; antibodies for GAPDH (sc-25778) and GST (sc-138) were from Santa Cruz Biotechnology (Santa Cruz, CA); the secondary antibodies, anti-rabbit IgG, HRP-linked antibody (#7074) and anti-mouse IgG, HRP-linked antibody (#7076) were purchased from CST (Danvers, MA).

Techniques: Expressing, Comparison, Quantitative Proteomics, Modification, Ubiquitin Proteomics

Journal: Nature Communications

Article Title: Role of SUMO activating enzyme in cancer stem cell maintenance and self-renewal

doi: 10.1038/ncomms12326

Figure Lengend Snippet: ( a ) Mass spectrometry identified TRIM21 as an Oct-1-interacting protein. Representative silver stain of HCT116 cells transfected with Flag-tagged Oct-1 or empty vector (Ctrl) for 2 days, lysed and immunoprecipitated with anti-Flag antibody-conjugated M2 beads. Proteins identified in each group are indicated in the graphic. ( b ) Representative IP-western blot analysis of the interaction between endogenous TRIM21 and Oct-1 in HT29 cells. ( c ) Knockdown of TRIM21 delayed Oct-1 degradation. Representative western blot of Oct-1 level over time in HT29 cells transfected with control non-silencing siRNA (siCtrl) or siTRIM21 for 3 days, followed by 100 μg ml −1 CHX treatment; GAPDH, loading control. Oct-1 decay curve (right panel) was determined by quantifying three independent experiments. ( d ) TRIM21 knockdown resulted in increased Oct-1 protein level and decreased ubiquitination of Oct-1. Representative western blot of HT29 cells transfected with siCtrl and siTRIM21 treated with or without 10 μM MG132 for 8 h. IP was carried out with cell lysates, and Oct-1, TRIM21 and ubiquitin were detected; GAPDH, loading control. ( e ) Overexpression of TRIM21 decreased Oct-1 and increased Oct-1 ubiquitination in cells. Representative western blot of HT29 cells were transfected with empty vector (Ctrl) or Flag-TRIM21 for 2 days, then treated with or without 10 μM MG132 for 8 h. IP was carried out with cell lysates, and western blot was performed to detect Oct-1, TRIM21 and ubiquitin; GAPDH, loading control.

Article Snippet: Specific antibodies to ALDH1A1 (1:1,000, 611194, BD Bioscience), SAE2 (1:1,000, ab58451, Abcam), SUMO-2,3 (1:500, M114-3, MBL), Ubc9 (1:1,000, #4918, Cell Signaling Technology, Inc.), Oct-1 (1:1,000, #8517, Cell Signaling Technology, Inc.), ubiquitin (1:1,000, MAB1510, Millipore), TRIM21 (1:1,000, sc-25351, Santa Cruz), IRF1 (1:1,000, #8478, Cell Signaling Technology, Inc.), SUMO-1 (1:500, #4930, Cell Signaling Technology, Inc.), SENP2 (1:1,000, sc-67075, Santa Cruz Biotech), PIAS1 (1:1,000, #3550, Cell Signaling Technology, Inc.) and GAPDH (1:1,000, sc-20357, Santa Cruz) were detected using the appropriate secondary antibodies (Licor) and visualized by Odyssey detection system (Licor).

Techniques: Mass Spectrometry, Silver Staining, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Over Expression

Journal: Nature Communications

Article Title: Role of SUMO activating enzyme in cancer stem cell maintenance and self-renewal

doi: 10.1038/ncomms12326

Figure Lengend Snippet: ( a ) Representative IHC staining indicates higher TRIM21 level corresponding with lower Oct-1 in shSAE2 group tumour tissue compared with shCtrl group (scale bar, 100 μm). Tumour tissues were from LDA assay described in . ( b ) Western blot (left) and quantification of SAE2, Oct-1, TRIM21 and ALDH levels in the same tumour tissues as a ; GAPDH, loading control. ‘1, 2 and 3' indicate tumour tissues from different mouse. ( c ) TRIM21 promoter activity was inhibited by overexpression of SAE2 and Ubc9 but increased by SENP1 overexpression as determined by luciferase reporter assay. HT29 cells were transfected with empty vector (Ctrl) or SAE2, UBC9 or SENP1 expression plasmid together with TRIM21 promoter luciferase reporter and Renilla plasmids. Dual-luciferase activity was measured after 48 h and normalized results were analysed with two-tailed Student's t -test. ( d ) TRIM21 mRNA level was suppressed by SAE2 or Ubc9 overexpression and enhanced with SENP1 overexpression in HT29 cells as determined by quantitative PCR (qPCR). ( e ) IRF1 SUMOylation site mutant K78R induced higher TRIM21 mRNA level than wild-type (WT) IRF1 as determined by qPCR. ( f ) TRIM21 protein level was suppressed on SAE2 or Ubc9 overexpression but enhanced with SENP1 overexpression as indicated by western blot; GAPDH, loading control. ( g ) Western blot showed overexpression of K78R mutant-induced higher TRIM21 protein level than WT IRF1 in shCtrl HT29 cells; GAPDH, loading control. * P <0.05, ** P <0.01 and *** P <0.001.

Article Snippet: Specific antibodies to ALDH1A1 (1:1,000, 611194, BD Bioscience), SAE2 (1:1,000, ab58451, Abcam), SUMO-2,3 (1:500, M114-3, MBL), Ubc9 (1:1,000, #4918, Cell Signaling Technology, Inc.), Oct-1 (1:1,000, #8517, Cell Signaling Technology, Inc.), ubiquitin (1:1,000, MAB1510, Millipore), TRIM21 (1:1,000, sc-25351, Santa Cruz), IRF1 (1:1,000, #8478, Cell Signaling Technology, Inc.), SUMO-1 (1:500, #4930, Cell Signaling Technology, Inc.), SENP2 (1:1,000, sc-67075, Santa Cruz Biotech), PIAS1 (1:1,000, #3550, Cell Signaling Technology, Inc.) and GAPDH (1:1,000, sc-20357, Santa Cruz) were detected using the appropriate secondary antibodies (Licor) and visualized by Odyssey detection system (Licor).

Techniques: Immunohistochemistry, Western Blot, Activity Assay, Over Expression, Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Expressing, Two Tailed Test, Real-time Polymerase Chain Reaction, Mutagenesis

Journal: Nature Communications

Article Title: Role of SUMO activating enzyme in cancer stem cell maintenance and self-renewal

doi: 10.1038/ncomms12326

Figure Lengend Snippet: ( a ) Overexpression of Oct-1 partially compensated for the reduction of CSC frequency by SAE2 knockdown in HT29 cells as determined by LDA. Stable cell lines were generated with lentivirus expressing pLenti CMV-hygro empty vector (EV) or pLenti CMV-Flag-Oct-1 in HT29 shCtrl and shSAE2 cells as shCtrl+EV, shSAE2+EV and shSAE2+Oct-1. ( b ) Representative western blot of the stable lines to confirm expression with Oct-1 and Flag-tag antibodies; GAPDH, loading control. ( c ) Overexpression of Oct-1 in SAE2 or Ubc9 knockdown cells restored ALDH + cells population in HT29 cells as measured by FACS analysis using the AldeFluor kit. HT29 cells were transfected with control non-targeting siRNA (SiCtrl), SAE2-targeting siRNA (SiSAE2) or Ubc9-targeting siRNA (SiUbc9) followed by Flag-Oct-1 plasmid or control empty vector transfection. After 3 days, cells were collected for FACS analysis using the AldeFluor kit. ( d ) Representative western blot of the samples from c ; quantification of the ALDH1A1 band intensity is shown on the bottom. ( e ) Knockdown of Ubc9 reduced CSC frequency, as shown by LDA using spheroid formation using HT29 stable cell lines expressing two different UBC9-targeting shRNA (shUBC9#1 and shUBC9#2). ( f ) Schematic diagram showing the mechanism of how SUMOylation regulates CSCs through Oct-1, TRIM21 and IRF1. * P <0.05, ** P <0.01 and *** P <0.001.

Article Snippet: Specific antibodies to ALDH1A1 (1:1,000, 611194, BD Bioscience), SAE2 (1:1,000, ab58451, Abcam), SUMO-2,3 (1:500, M114-3, MBL), Ubc9 (1:1,000, #4918, Cell Signaling Technology, Inc.), Oct-1 (1:1,000, #8517, Cell Signaling Technology, Inc.), ubiquitin (1:1,000, MAB1510, Millipore), TRIM21 (1:1,000, sc-25351, Santa Cruz), IRF1 (1:1,000, #8478, Cell Signaling Technology, Inc.), SUMO-1 (1:500, #4930, Cell Signaling Technology, Inc.), SENP2 (1:1,000, sc-67075, Santa Cruz Biotech), PIAS1 (1:1,000, #3550, Cell Signaling Technology, Inc.) and GAPDH (1:1,000, sc-20357, Santa Cruz) were detected using the appropriate secondary antibodies (Licor) and visualized by Odyssey detection system (Licor).

Techniques: Over Expression, Stable Transfection, Generated, Expressing, Plasmid Preparation, Western Blot, FLAG-tag, Transfection, shRNA

Journal: International Journal of Oncology

Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

doi: 10.3892/ijo.2015.3169

Figure Lengend Snippet: TRIM21 domain. (A) (Left) Vector map of the used pMK-RQ vector (Life Technologies), (right) amino acid sequence of TRIM21 and the sequences of an individual domains. (B) Western blot analysis after native 12.5% polyacrylamide gel. Detection of TRIM21 with Li-Cor 800 anti-goat, detection of LFG with Li-Cor-680 anti-rabbit. Sample 1a (PRY domain), sample 1b (SPRY domain), sample 2 (coiled-coil domain), sample 3 (B-box domain), sample 4 (RING domain), marker (M). The samples 2–4 show clearly visible bands of ~80 and 60 kDa. The bands at 80 kDa are larger but slightly less visible as the bands at the height of 60 kDa. In samples 1a and 1b no bands are visible.

Article Snippet: To identify the essential domain for the interaction between TRIM21 and LFG, different vectors, using pCMV6-AC-GFP vector (NM_003141; OriGene, Cambridge, UK) were cloned containing the sequences of TRIM21 with the deletion of an entire domain.

Techniques: Plasmid Preparation, Sequencing, Western Blot, Marker

Journal: International Journal of Oncology

Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

doi: 10.3892/ijo.2015.3169

Figure Lengend Snippet: Analysis of the interaction. Array analysis of the interaction of over 9,000 different proteins with LFG. Recombinant LFG protein (10 μM) was used and detected by specific first antibody (FAIM2; Santa Cruz Biotechnology, Inc.) and fluorescence labeled second antibody (Alexa Fluor 546). The display window shows the signal stimulated by the interaction of LFG with TRIM21 (white signal).

Article Snippet: To identify the essential domain for the interaction between TRIM21 and LFG, different vectors, using pCMV6-AC-GFP vector (NM_003141; OriGene, Cambridge, UK) were cloned containing the sequences of TRIM21 with the deletion of an entire domain.

Techniques: Recombinant, Fluorescence, Labeling

Journal: International Journal of Oncology

Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

doi: 10.3892/ijo.2015.3169

Figure Lengend Snippet: Z-score of protein-protein interaction array-analysis.

Article Snippet: To identify the essential domain for the interaction between TRIM21 and LFG, different vectors, using pCMV6-AC-GFP vector (NM_003141; OriGene, Cambridge, UK) were cloned containing the sequences of TRIM21 with the deletion of an entire domain.

Techniques: Variant Assay, Activation Assay

Journal: International Journal of Oncology

Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

doi: 10.3892/ijo.2015.3169

Figure Lengend Snippet: Co-immunoprecipitation. (A) Native western blot analysis after co-immunoprecipitation of LFG and TRIM21. LFG was specifically isolated from the cell lysate of MDA-MB-231 after transfection with vectors coding for LFG and TRIM21 (24 h) by use of antibody coated magnetic beads (Dynabeads; Invitrogen). Detection of LFG (FAIM2) and TRIM21 (Ssa1/2) (both from Santa Cruz Biotechnology, Inc.) by specific first antibodies and fluorescence labeled second antibodies. LFG is visible as a green signal (Alexa Fluor 546), TRIM21 is visible as a red signal (Alexa Fluor 488), and combined signals are visible as a yellow signal. (B) Analysis of LFG and TRIM21 protein in the MDA-MB-231 breast cancer cells 24 h after transfection with vectors coding for LFG and TRIM21, by specific first and fluorescence labeled second antibodies. a, Green signal for LFG; b, red signal for TRIM21; c, blue signal for DAPI-stained core; d, overlay of the single signals with yellow signals in places where green and red signals appear in the same spot.

Article Snippet: To identify the essential domain for the interaction between TRIM21 and LFG, different vectors, using pCMV6-AC-GFP vector (NM_003141; OriGene, Cambridge, UK) were cloned containing the sequences of TRIM21 with the deletion of an entire domain.

Techniques: Immunoprecipitation, Western Blot, Isolation, Transfection, Magnetic Beads, Fluorescence, Labeling, Staining

Journal: International Journal of Oncology

Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

doi: 10.3892/ijo.2015.3169

Figure Lengend Snippet: Trim21 and LFG expression. (A) Results of real-time PCR-analysis of Trim21-expression after 24 h under different culture conditions. Mock, Untreated MDA-MB-231 as negative control, Trim21, 2.0 μl recombinant human Trim21 protein added to culture medium; siControl, adenoviral transfection of MDA-MB-231 with empty vector; siLFG, adenoviral transfection of MDA-MB-231 with vector coding for siRNA against LFG. (B) Expression analysis of LFG protein under different culture conditions by western blot analysis using 25 μg of total protein. a, Adenoviral transfection of MDA-MB-231 with vector coding for siRNA against LFG (24 h). b, Adenoviral transfection of MDA-MB-231 with empty vector. c, Untreated MDA-MB-231 as negative control. d, MDA-MB231 cultivated with 2.0 μg recombinant Trim21-protein in culture medium.

Article Snippet: To identify the essential domain for the interaction between TRIM21 and LFG, different vectors, using pCMV6-AC-GFP vector (NM_003141; OriGene, Cambridge, UK) were cloned containing the sequences of TRIM21 with the deletion of an entire domain.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Negative Control, Recombinant, Transfection, Plasmid Preparation, Western Blot

Journal: International Journal of Oncology

Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

doi: 10.3892/ijo.2015.3169

Figure Lengend Snippet: Caspase-3-activity and cell cycle-analysis. (A) Analysis of caspase-3-activity in MDA-MB-231 cells. Cultivation for 48 h under different conditions and addition of 100 ng agonistic anti-Fas (clone CH11; Abcam). Measurement of caspase-3-activity after 24 h. TRIM21, addition of 1.0 μg/ml recombinant human TRIM21-protein every 24 h. Control, untreated MDA-MB-231 cultivated for 48 h. Detection of LFG, TRIM21 and actin by specific first antibodies and fluorescence labeled second antibodies. (B) Plots of cell cycle-analysis of MDA-MB-231 after 24 h cultivation in presence of different concentration of recombinant human TRIM21-protein after staining with propidium iodide. a-1, Cultivation of MDA-MB-231 without addition of TRIM21-recombinant protein. b-1, Addition of 0.25 μg/ml TRIM21-recombinant protein to the culture medium. c-1, Addition of 0.5 μg/ml TRIM21-recombinant protein to the culture medium. d-1, Addition of 0.75 μg/ml TRIM21-recombinant protein to the culture medium. e-1, Addition of 1.0 μg/ml TRIM21-recombinant protein to the culture medium.

Article Snippet: To identify the essential domain for the interaction between TRIM21 and LFG, different vectors, using pCMV6-AC-GFP vector (NM_003141; OriGene, Cambridge, UK) were cloned containing the sequences of TRIM21 with the deletion of an entire domain.

Techniques: Activity Assay, Cell Cycle Assay, Recombinant, Fluorescence, Labeling, Concentration Assay, Staining

Journal: International Journal of Oncology

Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

doi: 10.3892/ijo.2015.3169

Figure Lengend Snippet: Arrays. (A) Tissue samples of breast tumors having different degrees of malignity (US Biomax, Inc.) (a and b). Specific antibody against Trim21 and fluorescence labeled second antibody (Li-Cor-800), and staining of nucleic acid with Syto-60. Detection was carried out using Odyssey (Li-Cor Biosciences). (B) Bar chart of the fluorescence intensity of the tissue samples, degrees of malignity (IIa, IIb, IIIa, IIIb and IV); * P<0.05 vs. control (NAT). NAT, native tissue sample. Benign, benign breast tumor. Inflammation, inflamed tissue sample.

Article Snippet: To identify the essential domain for the interaction between TRIM21 and LFG, different vectors, using pCMV6-AC-GFP vector (NM_003141; OriGene, Cambridge, UK) were cloned containing the sequences of TRIM21 with the deletion of an entire domain.

Techniques: Fluorescence, Labeling, Staining

Journal: International Journal of Oncology

Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

doi: 10.3892/ijo.2015.3169

Figure Lengend Snippet: NF-κB array. Bar chart of gene expression of common NF-κB regulated genes after real-time PCR-analysis. Trim21, MDA-MB-231 cultivated with 2.0 μg/ml recombinant human Trim21-protein for 24 h. Control, MDA-MB-231 cells cultivated for 24 h under standard conditions.

Article Snippet: To identify the essential domain for the interaction between TRIM21 and LFG, different vectors, using pCMV6-AC-GFP vector (NM_003141; OriGene, Cambridge, UK) were cloned containing the sequences of TRIM21 with the deletion of an entire domain.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Recombinant