RESUMEN

OBJECTIVE: To study the expression of recombinant human SCF-TPO fusion protein and its biological function. METHODS: Four primers were designed according to known sequences of TPO and SCF. The functional amino acid domains of TPO and SCF were amplified by RT-PCR from fetus hepatocytes, respectively. The expression plasmid pET32a/SCF-TPO was constructed by VOE gene fusion technique and expressed in E. coli BL21(DE3) plysS as inclusion body after isopropyl-beta-D-1-thiogalactopyranoside (IPTG) induction. The fusion protein was tested by SDS-PAGE and Western blot. The biological functions of SCF-TPO fusion protein in MO7e cells was investigated by MTT method after purification with metal chelating chromatography. RESULTS: The high expression SCF-TPO fusion protein was obtained, reaching up to 30% of the total cellular protein. Western blot verified the correct fusion expression and MTT results showed the growth promoting effect of the SCF-TPO fusion protein on MO7e cells, with a higher promoting activity at 100 ng/ml. CONCLUSIONS: Expressed SCF-TPO fusion protein after renaturation has biological activity in promoting the proliferation of MO7e cells.

Asunto(s)

Proliferación Celular , Proteínas Recombinantes de Fusión/genética , Factor de Células Madre/genética , Trombopoyetina/genética , Western Blotting , Línea Celular Tumoral , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Vectores Genéticos , Humanos , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Células Madre/metabolismo , Factor de Células Madre/fisiología , Trombopoyetina/metabolismo , Trombopoyetina/fisiología

RESUMEN

AIM: Design, construction, expression in E coli and protein characteristics prediction of bimolecular thrombopoietin (T-T) with more stability, efficiency, and lower toxicity. METHODS: The expression vectors of TPO and T-T, pET32 a(+)/TPO and pET32 a (+)/T-T, had been constructed by molecular cloning methods. Then, they were expressed in host bacterium. Their products were identified by Western blot. The protein characteristics, such as second structure, antigenicity, hydrophilicity, flexibility and isoelectric point, were predicted by DS Gene and Protscale software. RESULTS: The expressing vectors pET32a(+)/TPO and T-T were constituted correctly and expressed in origami (DE3), and their expression efficiency were more than 40 percent of total protein. T-T was identified correctly by Western blot. DS Gene and Protscale software predict the protein characteristics of TPO sequences in T-T molecule were no change, there was high flexibility in the linker domain. But two amino acids in T-T molecule have been mutated, and an insert fragment with 34 amino acids following the linker had antigenicity, hydrophilicity, and beta-sheet structure. CONCLUSION: We have constructed correctly and expressed T-T with high level in E Coli. Protein characteristics prediction of T-T accords with our design.

Asunto(s)

Vectores Genéticos , Proteínas Recombinantes de Fusión/genética , Trombopoyetina/genética , Clonación Molecular , Escherichia coli , Expresión Génica

RESUMEN

The objective was to identify some biochemical and physical properties for fusion protein IL6D24-PE40KDEL. Edman degradation, SDS-PAGE, peptide mass fingerprinting, Western blot and MTT were used for identification of the protein. The results showed that the sequence of N-terminus is Met-Ile-Asp-Lys-Gln-Ile, Met was added because of prokaryotic expression system; Western blot revealed that the purified protein could react with IL6 and PEA antibody. The purified protein IL6D24-PE40KDEL could kill the multiple myeloma cell lines U266 expressing high affinity IL6R, but it could not kill the cell lines CEM which not expressed IL6R; The molecular weight was 58.7 kD measuring by SDS-PAGE; peptide mass fingerprinting (PMF) confirmed that the construction of IL6D24-PE40KDEL was correct. A novel protein by Peptident database in EXPASY web site was identified. In conclusion, IL6D24-PE40KDEL is a new targeting protein with bioactivity of specific killing effect.

Asunto(s)

ADP Ribosa Transferasas/metabolismo , Exotoxinas/metabolismo , Interleucina-6/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , ADP Ribosa Transferasas/química , ADP Ribosa Transferasas/farmacología , Secuencia de Aminoácidos , Western Blotting , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Exotoxinas/química , Exotoxinas/farmacología , Humanos , Interleucina-6/química , Interleucina-6/farmacología , Datos de Secuencia Molecular , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/farmacología , Análisis de Secuencia de Proteína , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción

RESUMEN

OBJECTIVE: A novel recombinant interleukin6-Pseudomonas exotoxin fusion protein, having targeted cytotoxicity for leukemic cells highly expressing IL6R has been molecularly designed and constructed in this study. METHODS: Firstly, REDLK at C-terminus of Pseudomonas exotoxin PE40 was replaced with KDEL using point mutagenesis technology. Secondly, a cDNA encoding interleukin-6 devoid of N-terminal 24 amino acids [IL6D24] was fused to 5' terminus of PE40KDEL DNA by the method of gene splicing by overlap extension, which could generate recombinant IL6D24-PE40KDEL and IL6D24-Linker-PE40KDEL fusion genes. Thirdly, recombinant fusion genes IL6D24-PE40KDEL and IL6D24-Linker-PE40KDEL were ligated into the EcoR I and Sma I cloning sites in the pBV220 plasmids respectively and then transformed into E.coli HB101 cells. The expressed recombinant exotoxin fusion proteins were purified to electrophoresis purity by Mono Q column chromatography. Its selectively killing was tested by the MTS colorimetric method using both U937 and CEM cells lines. RESULTS: Recombinant exotoxin fusion proteins IL6D24-PE40KDEL was expressed as a form of inclusion bodies at higher level of 40% approximately of total proteins in bacterial cells. Western blot showed that the purified products could react specifically with IL6 monoclonal antibody and PEA antiserum, respectively. IL6D24-PE40KDEL was selectively cytotoxic to U937 cells expressing IL6R-positive with ID50 of 250 ng/ml, and but not CEM cells expressing IL6R-negative. CONCLUSIONS: Two novel recombinant interleukin6-Pseudomonas exotoxin fusion proteins IL6D24-PE40KDEL and IL6D24-Linker-PE40KDEL have been successfully constructed and they can selectively kill the leukemic cells expressing highly IL6R.

Asunto(s)

Proteínas Bacterianas/química , Citotoxicidad Inmunológica , Exotoxinas/química , Interleucina-6/química , Interleucina-6/farmacología , Leucemia de Células T/inmunología , Proteínas Recombinantes de Fusión/química , ADP Ribosa Transferasas , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Anticuerpos Antineoplásicos/biosíntesis , Anticuerpos Antineoplásicos/genética , Antígenos de Neoplasias/biosíntesis , Antígenos de Neoplasias/inmunología , Proteínas Bacterianas/aislamiento & purificación , Línea Celular Tumoral , Clonación Molecular , Escherichia coli/genética , Exotoxinas/genética , Exotoxinas/aislamiento & purificación , Humanos , Interleucina-6/aislamiento & purificación , Leucemia de Células T/patología , Plásmidos/genética , Receptores de Interleucina-6/biosíntesis , Receptores de Interleucina-6/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Células U937

RESUMEN

An inevitable trend for the development of new treatment of leukemia is to use targeted strategy. IL-6/IL-6R is important one of the cytokine receptor targeted treatment systems. Many malignant cells, including multiple myeloma, prostate carcinoma and leukemia etc., have been shown to express IL-6R. Leukemia cells, especialy in U937, TF1, KG1 cell lines highly express the high affinity IL-6R. IL-6 recombinant toxin is cytotoxic in vitro to leukemia cell expressing high affinity IL-6R; in vivo the fusion toxin can result the reduction of leukemic cell load in animal leukemia model but have no effect on normal hematopoiesis in non-leukemic animal. On the basis of these pre-clinical studies, IL-6 recombinant toxin my become novel drug for the treatment of leukemia and cancer in future.