https://febs.onlinelibrary.wiley.com/doi/pdf/10.1002/1878-0261.12850

High‐throughput proteomics of breast cancer interstitial fluid: identification of tumor subtype‐specific serologically relevant biomarkers

C35 is a novel tumor biomarker associated with metastasis progression. To investigate the interaction factors of C35 in its high expressed breast cancer cell lines, we constructed bait recombinant plasmids of C35 gene and T47D cell cDNA library for yeast two-hybrid screening. Full length C35 sequences were subcloned using RT-PCR from cDNA template extracted from T47D cells. Based on functional domain analysis, the full-length C351-348bp was also truncated into two fragments C351-153bp and C35154-348bp to avoid auto-activation. The three kinds of C35 genes were successfully amplified and inserted into pGBKT7 to construct bait recombinant plasmids pGBKT7-C351-348bp, pGBKT7-C351-153bp and pGBKT7-C35154-348bp, then transformed into Y187 yeast cells by the lithium acetate method. Auto-activation and toxicity of C35 baits were detected using nutritional deficient medium and X-α-Gal assays. The T47D cell ds cDNA was generated by SMARTTM technology and the library was constructed using in vivo recombination-mediated cloning in the AH109 yeast strain using a pGADT7-Rec plasmid. The transformed Y187/pGBKT7-C351-348bp line was intensively inhibited while the truncated Y187/pGBKT7-C35 lines had no auto-activation and toxicity in yeast cells. The titer of established cDNA library was 2 × 107 pfu/mL with high transformation efficiency of 1.4 × 106, and the insert size of ds cDNA was distributed homogeneously between 0.5-2.0 kb. Our research generated a T47D cell cDNA library with high titer, and the constructed two C35 "baits" contained a respective functional immunoreceptor tyrosine based activation motif (ITAM) and the conserved last four amino acids Cys-Ile-Leu-Val (CILV) motif, and therefore laid a foundation for screening the C35 interaction factors in a BC cell line.

Construction of C35 gene bait recombinants and T47D cell cDNA library.

La proteína 7 unida al receptor del factor de crecimiento (GRB7) en cáncer de mama

Tumor metastasis is a sequential event accounting for numerous cancer-related fatalities worldwide. The process of metastasis serially involves invasion, intravasation, extravasation, and tumor growth at the secondary site. Migration and invasion enhancer 1 (MIEN1) is a membrane associated protein overexpressed in various human cancers. Biological activity of MIEN1 is driven by geranylgeranyltransferase-I mediated prenylation at CAAX motif and methylation of the prenylated protein that anchors MIEN1 into the cellular membrane. Post-translationally modified MIEN1 interacts with Syk kinase and Annexin A2 protein; polymerizes G-actin and stabilizes F-actin filament; induces focal adhesion kinase phosphorylation and decrease cofilin phosphorylation implicated in both invasion and metastasis of different cancer types. In the present review, we discuss the structure, function, and involvement of MIEN1 in cancer progression. We also highlight the future prospects of MIEN1 as an emerging molecule and novel target in cancer cell invasion and metastasis.

/pdf/emerging-role-of-migration-and-invasion-enhancer-1-mien1-in-1gonp269fi.pdf

Emerging Role of Migration and Invasion Enhancer 1 (MIEN1) in Cancer Progression and Metastasis

To investigate C35 protein expression in breast carcinoma and to investigate its clinicopathological significance, a total of 68 cases of breast carcinoma and 20 cases of normal breast tissue samples were obtained from the clinic. Protein expression of C35, ER, PR and HER-2 were determined using immunohistochemistry. The correlations between C35 expression and clinicopathological parameters were analyzed on the basis of individual clinicopathologic records. Overexpression of C35 was detected in 56 of 68 (82.35%) breast carcinoma samples and only 3 of 20 (15%) normal breast tissue samples, and frequency of C35 expression was significantly associated with clinical Tumor Node Metastasis staging and Scarff-Bloom-Richardson grade (p < 0.05), but was not related to patients age, menstrual status and tumor diameter. C35 expression was positively related with the expression of HER-2 (r = 0.207), whereas negatively related with the expression of ER and PR. Further, C35 was prevalent in all four molecular subtypes of breast carcinoma with no significant difference of expression frequency. However, they have significant differences in lymphatic metastasis cases compared to the non-metastasis cases (p < 0.05). Since C35 protein was extensively expressed in all stages of breast carcinoma, and was closely associated with tumor progression and lymph node metastasis, it might be used as a reliable biomarker or therapeutic target for diagnosis and treatment.

Overexpression of C35 in breast carcinomas is associated with tumor progression and lymphnode metastasis.

Objective: To express and purify the Toxoplasma gondii RH strain immune mapped protein 1 (IMP1) protein through the prokaryotic expression system, and to identify the purified protein. Method: Gene of TgIMP1 was attained by RT-PCR and the amplified product was identified by TA-clone and sequencing. The identified fragment of IMP1 was connected into the expression vector pET28b and the recombinant pET28b-TgIMP1 was identified by double digestion and sequencing. The fusion protein TgIMP1 with 6 × His tag was expressed in E. coli BL21 (DE3) host and the optimal inducing conditions were investigated by adjusting the harvesting time interval, inducing temperature and concentration of isopropyl β-D-thiogalactoside (IPTG). The solubility detection of TgIMP1 protein and the massive purification of it were conducted with Ni2+-affinity purification. The purified product was identified by Coomassie-stained SDS-PAGE and immune-blotting. Results: The prokaryotic expression recombinant pET28bTgIMP1 was successfully established on the basis of the identified TgIMP1 gene by TA-clone. Recombinant proteins of TgIMP1 could be efficaciously and largely expressed in E. coli BL21 strain with 0.3 mM IPTG for 9 h at 20°C, and with the characteristics of good stability, high solubility and purity, and fine immunity. Conclusion: TgIMP1 protein could be expressed in the prokaryotic host strain with satisfied stability and solubility. Our findings may lay the foundation for the future protein-based investigations such as development of subunit vaccine and structural determination.

Purification and characterization of Toxoplasma gondii immune mapped protein-1

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Overexpression of C35 in breast carcinomas is associated with tumor progression and lymphnode metastasis.

Purification and characterization of Toxoplasma gondii immune mapped protein-1