The Construction And Expression Of A Novel Recombinant Protein TAT/54R/KDEL And Characterization Of Its CXCR4 Phenotypic Knockout Function

It has been shown that interaction between CXCR4 (CXC chemokine receptor 4) and its specific ligand SDF-1(Stromal cell-derived factor-1) plays an important role in the metastasis of many malignant tumors. Therefore, it may be of great significance to effectively prevent tumor metastasis by targeting CXCR4 and blocking interaction between CXCR4 and SDF-1. Therefore, a competitive antagonist of CXCR4, named SDF-1/54R, was constructed by gene reconstruction of SDF-1 in our previous research, and it has been proved that SDF-1/54R could eliminate CXCR4 on cell surface by induction of internalization of CXCR4 quickly, however, the inhibitory effect of SDF-1/54R on CXCR4 induced cell migration was also temporary because of the re-expression of internalized CXCR4 receptor on cell membrane. Accordingly, a new approach to permanently inhibit CXCR4 expression is necessary in order to obtain a clinical valuable CXCR4 antagon. The intrakine phaenotype knockout technology will be a new strategy to block CXCR4 expression on tumor cell surface permanently due to the properties of intrakine such as low molecular weight, strong specificity and high targetting. Above on this, eukaryotic vector and adenovirus vector of intrakine, SDF-1/54R/KDEL, were constructed by linking SDF-1/54R and an endoplasmic reticulum (ER) retention signal segment (KDEL) together. It has been demonstrated that expression of CXCR4 on MOLT-4 cell which transfected by SDF-1/54R/KDEL was reduced dramatically and a permanent inhibition of migration was observed. Allow for the multiplicity of transgenic technology operation and the low safety ensurance in systemic application, a further strategy of delivering chemotatic factor intracellularly to knockout CXCR4 on cell surface was necessary. Therefore, based on the newest research of transmembrane peptide, the second gene reconstruction of SDF-1/54R/KDEL was carried on by linking a highly effective transmembrane vector TAT(47～57) to its N-extremity. The high transmembrane effect of TAT and the ER retention function of KDEL both help to introduce fusion protein SDF-1/54R into ER of tumor cells in order to specifically bind to the newly-synthetic target receptor CXCR4 intracellularly, accordingly block the expression of CXCR4 on cell surface and consequently the following tumor metastasis signaling mediated by CXCR4/SDF-1 interaction. The results will provide direct experimental evidence to investigate the feasibility and effectivity of the new strategy which exploring CXCR4 as a target in cancer gene therapy.Objectives:A new chimeric protein: TAT/54R/KDEL was constructed by the second gene reconstruction of SDF-1/54R, via a highly effective transmembrane vector TAT(47～57) and the endoplasmic reticulum (ER) retention signal (KDEL) linked to each extremity of SDF-1/54R respectively, and the inhibition of tumor metastasis was tested both in vitro and in vivo.Methods:①GFP/KDEL and 54R/KDEL gene were obtained from GFP/pET-28a(+) and 54R/pET-28a(+) constructed previously with PCR, and linked with KDEL gene fragment respectively. Then the two part were inserted into the downstream MCS of TAT in prokaryotic expression vector pTAT-HA respectively to obtain the recombinant plasmids TAT/GFP/KDEL/pTAT-HA and TAT/54R/KDEL/pTAT-HA .②The identificated recombinant plasmids TAT/GFP/KDEL/pTAT-HA and TAT/54R/KDEL/pTAT-HA were subsequently transformed into E coli expression strain BL21(DE3), the expression of recombinant protein was induced by 0.5μM IPTG. and verificated by Western blot. Then a Ni2+ affinity chromatography column was used to purificate recombinant protein by the 6×His-Tag at the N-extremity of the protein. Active TAT/GFP/KDEL could be obtained just by dislysis and concentration due to its soluble expression property, however, TAT/54R/KDELL needs deliquation, dislysis and ultrafiltration for renaturation as it exists in inclusion body.③MOLT-4 cell line was used to test the transmembrane and ER retention capability of TAT/GFP/KDEL to determine the feasibility of designed proposal initially. The bioactivity of TAT/54R/KDEL as transmembrane capability and targeting property was detected by fluorescence microscope and fluorescence microplate reader on MOLT-4 cells which have an overexpression of CXCR4 and CNE2 cells which are CXCR4 negative. Laser confocal microscopy was used to detect intracellular location of TAT/54R/KDEL on ER. It is of great importance to test CXCR4 knockout effect of TAT/54R/KDEL, so a flow cytometer was used firstly to detect influence of TAT/54R/KDEL on CXCR4 expression on cell surface, then a chemotaxis essay to investigate TAT/54R/KDEL's inhibitory effect on MOLT-4 cell migration and finally Western blot was applied to evaluate the effectiveness of TAT/54R/KDEL on CXCR4 knockout on cell surface. Besides, trypan blue rejection experiment and MTT method was used to test cytotoxicity and influence on cell proliferation of TAT/54R/KDEL. ④The animal model of breast cancer metastasis was established by transplanting 4T1 cell into the second breast subcutaneous fat pad of BALB/c mice to detect the inhibitory effect of TAT/54R/KDEL on development and metastasis of the transplanted tumor in tumor-bearing mice. RT-PCR and flow cytometer was applied to detect variation of CXCR4 and SDF-1 expression in primary tumor and metastatic tumor in order to investigate the interaction of metastasis inhibition role of TAT/54R/KDEL and its knockout effect on CXCR4, and illuminate the action mechanism of TAT/54R/KDEL in vivo.Results:①GFP/KDEL and 54R/KDEL gene were obtained successfully with PCR from GFP/pET-28a(+) and 54R/pET-28a(+) constructed previously and inserted into pTAT-HA vector. Both were confirmed to be correct by PCR, enzyme digestion and gene sequencing.②Overexpression of TAT/GFP/KDEL/pTAT-HA and TAT/54R/KDEL/pTAT-HA in BL21 was confirmed by Western blot. Purified target proteins TAT/GFP/KDEL and TAT/54R/KDEL (>95%) were obtained by nickel affinity chromatography and HPLC purification. The TAT/54R/KDEL in inclusion body was renaturated by dilution, dislysis and ultrafiltration.③High transmembrane effectiveness and capability of exact ER retention of TAT/GFP/KDEL in MOLT-4 cells was verified by fluorescence microscope and fluorescence microplate reader, which offering a initial feasibility of the designed proposal. When compared with CXCR4- CNE2 cells, TAT/54R/KDEL showed a better transmembrane and targeting capability, and exact located on ER in MOLT-4 cells on which CXCR4 is overexpressed, which established the foundation of CXCR4 phenotype knockout. Flow cytometry proved that TAT/54R/KDEL had a permanent CXCR4 phenotype knockout effect on MOLT-4 cells. Chemotaxis essay showed that TAT/54R/KDEL could restrain SDF-1 induced cell migration effectively and Western blot demonstrated that the CXCR4 phenotype knockout effectiveness of TAT/54R/KDEL was more thoroughly compared with SDF-1/54. Trypan blue rejection experiment and MTT method confirmed that TAT/54R/KDEL has a proliferation inhibitory effect on MOLT-4 cells which has no effect on CNE2 cells.④The animal model of breast cancer metastasis was established successfully by transplanting 4T1 cell into the second breast subcutaneous fat pad of BALB/c mice, and a dramatic inhibitory effect of TAT/54R/KDEL on development and metastasis of the transplanted tumor was detected. The correlation breast tumor metastasis with CXCR4 expression on tumor cell surface and SDF-1 expression in metastatic tumor tissue was confirmed to be close by RT-PCR and flow cytometry. TAT/54R/KDEL has a inhibition role on breast cancer metastasis to some extent via its CXCR4 knockout effect.Conclusion:①E.Coli expression system of TAT/GFP/KDEL and TAT/54R/KDEL was constructed successfully and bioactive TAT/GFP/KDEL and TAT/54R/KDEL proteins were obtained by expression, separation and purification.②High transmembrane effectiveness and capability of exact ER retention of TAT/GFP/KDEL on MOLT-4 cells were verified, confirming the feasibility of the designed proposal initially.③TAT/54R/KDEL has a permanent CXCR4 phenotype knockout effect on MOLT-4 cell surface in vitro.④The animal model of breast cancer metastasis was established successfully by transplanting 4T1 cell into the breast subcutaneous fat pad of BALB/c mice.⑤The inhibition role of TAT/54R/KDEL on breast cancer metastasis was confirmed by experiment in vivo to some extent.