High expression of Fightless I (FLII) is associated to multiple tumors. Based on our previous study that FLII might be involved in the nuclear export, we assessed the possible interaction of FLII with the nuclear envelop associating proteins Importin β and Nup88. We first constructed GST-FLII, GST-LRR recombinant plasmids and transformed them into the Rosetta strain to produce GST-FLII, GST-LRR fusion protein. After purification of these proteins, GST-pull down, as well as co-immunoprecipitation, were used to test the interaction of FLII with Importin β and Nup88. FLII interacted with Importin β and Nup88, and FLII LRR domain is responsible for these interactions. Thus, FLII may play a role in nuclear export through interaction with Importin β and Nup88.
Humans ; Microfilament Proteins ; metabolism ; Nuclear Pore Complex Proteins ; metabolism ; Receptors, Cytoplasmic and Nuclear ; metabolism ; Recombinant Fusion Proteins ; metabolism ; beta Karyopherins ; metabolism

The cDNA of cattle Ghrelin gene was amplified from abomasum fundic gland mRNA of Qinchuan Cattle by RT-PCR. PCR product was cloned into the T vector pGM-T to construct pGh-T1 for sequencing. Then the cDNA was subcloned into the prokaryotic expressing plasmid vector pET32a (+) and transformed into host Escherichia coli strain BL21 (DE3) for expression. The expression of pGh-32 mature Ghrelin protein was induced by IPTG and was identified by SDS-PAGE. The expression product was observed with soluble protein and inclusion body. Western blotting showed that the recombinant protein was recognized by his-antibody specifically. The protein was purified by Ni-NTA column and was used to inject rabbits to obtain polyclona antibody. ELISA result showed that the antibody titer was 1:12 800. The immunohistochemistry test between the hypothalamus arcuate nucleus and the antibody showed that fusion protein had biological activity. This will provide a basis for further study on the biological function of Ghrelin protein to growth and development and fat deposition of cattle.
Animals ; Cattle ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Ghrelin ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism

To express Pleurocidin in Escherichia coli and to enhance the secretory efficiency of the fusion protein, the gene encoding Pleurocidin was ligated with Cherry DNA sequence via blunt-end ligation. Then this fusion gene was cloned into pET22b (+) vector and the recombinant plasmid was transformed into E. coli BL21 (DE3). Lactose was used to induce expression of fusion protein. The recombinant plasmid pET22b (+) -CP was successfully constructed and high-level expression of fusion protein was induced with lactose. Statistics showed that addition of glycine after 16 h of induction significantly enhanced the secretory efficiency of the fusion protein. After hydrolysis of the fusion protein by diluted hydrochloric acid and some further purification steps, r-Pleurocidin was obtained with antibacterial activity against E. coli DH5α and Bacillus subtilis BS168. In conclusion, the fusion protein was expressed in E. coli and biologically active r-Pleurocidin was obtained after hydrochloric acid cleavage and purification.
Animals ; Cloning, Molecular ; Escherichia coli ; metabolism ; Fish Proteins ; biosynthesis ; Flounder ; Recombinant Fusion Proteins ; biosynthesis

Self-assembling amphipathic peptides (SAPs) have alternating hydrophilic and hydrophobic residues and can affect the thermal stabilities and catalytic properties of the fused enzymes. In this study, a novel multifunctional tag, S1vw (HNANARARHNANARARHNANARARHNARARAR) was developed to modify fused enzymes. After fusing S1vw at the enzymes/proteins N-terminus through a PT-linker, the crude enzymatic activities of polygalacturonate lyase and lipoxygenase were enhanced 3.1- and 1.89-fold, respectively, compared to the wild-type proteins. The relative fluorescence intensity of the green fluorescent protein was enhanced 16.22-fold. All the three S1vw fusions could be purified by nickel column with high purities and acceptable recovery rates. Moreover, S1vw also induced the thermostabilities enhancement of the fusions, with polygalacturonate lyase and lipoxygenase fusions exhibiting 2.16- and 3.2-fold increase compared with the corresponding wild-type, respectively. In addition, S1vw could enhance the production yield of green fluorescent protein in Escherichia coli and Bacillus subtilis while the production of GFP and its S1vw fusion changed slightly in Pichia pastoris. These results indicated that S1vw could be used as a multifunctional tag to benefit the production, thermal stability and purification of the fusion protein in prokaryotic expression system.
Escherichia coli ; Green Fluorescent Proteins ; Hydrophobic and Hydrophilic Interactions ; Peptides ; Pichia ; Recombinant Fusion Proteins ; metabolism

Resveratrol is a natural phytoalexin with special pharmacological and health functions. Stilbene synthase (STS) is a key and rate-limiting enzyme in the biosynthesis of resveratrol that is present only in a limited number of plants. The content of resveratrol from Polygonum cuspidatum is more than 1000 times higher than grapes and peanuts. We speculate that the catalytic ability of different STS may be one of the reasons causing differences in the content of resveratrol. To verify the above speculation, Vitis vinifera stilbene synthase gene (VvSTS) was amplified according to overlap PCR protocol with genomic DNA as template. VvSTS and PcSTS (PcPKS5) were analyzed through heterologous expression in Escherichia coli. The expression products were purified with Ni-NTA sepharose affinity chromatography and desalted through PD-10 column. The molecular weight of the two fusion proteins was about 43 kDa. Enzyme reaction and product analysis showed that the two products were resveratrol. The enzyme kinetic analysis showed that the catalyze efficiency (Kcat/Km) of PcPKS5 was 2.4 times of the VvSTS. Our findings confirms that STS from certain plants has much higher catalytic capability.
Acyltransferases ; metabolism ; Fallopia japonica ; enzymology ; Recombinant Fusion Proteins ; biosynthesis ; Stilbenes ; metabolism ; Vitis ; enzymology

The fused gene (PTH-TFN) of parathyroid hormone (PTH) gene and transferring N-terminal half-molecule (TFN) gene was amplified by multiple PCR and inserted into pPIC9 vector. The recombinant plasmid pPIC9-PTH-TFN was transformed into Pichia pastoris GS115 by PEG. After methanol induction, the target protein was expressed in fermentation supernatant at high level. The fused protein PTH-TFN with purity being higher than 95% was finally obtained after purification through two-step chromatography: SP Sepharose Fast Flow and Phenyl Sepharose Fast Flow. Western blot analysis and adenylate cyclase assay proved that the fused protein exhibited the bioactivity to stimulate cAMP synthesis and the ability to bind Fe3+ in the Fe3+ saturation study as the recombinant TFN did indicating that TFN could be used as the transcellar carrier of PTH.
Artificial Gene Fusion ; Cloning, Molecular ; Humans ; Parathyroid Hormone ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transferrin ; genetics

Due to their potential application as novel antibiotics, antimicrobial peptides are attracting much attention. Large quantities of highly purified peptides are crucial to basic and clinical studies. Natural resources of antimicrobial peptides are limited and hard to purify, chemical synthesis is of high-cost and unstable, so recombinant expression of antimicrobial peptides is a cost-effective way. Escherichia coli has been the most widely used as host to express antimicrobial peptides with fusion protein, which can not only avoid the lethal effect towards the host, but also protect the peptide from degradation by proteases. Combined with our research, the present article reviews the progress of fusion vector, cleavage methods and optimization options for antimicrobial peptides production with fusion protein in Escherichia coli.
Antimicrobial Cationic Peptides ; biosynthesis ; Escherichia coli ; metabolism ; Genetic Vectors ; Recombinant Fusion Proteins ; biosynthesis

Ubiquitin-ribosomal protein S27a(UBRPS27a) is a fusion protein of Ubiquitin and ribosomal protein. The N-terminal is ubiquitin and C-termina is ribosomal protein S27a with a high conservative zinc finger domain of the C2-C2 type. When it was expressed in eukaryotes,The intact fusion protein were rapidly processed to free ubiquitin monomer and ribosomal protein S27a (RPS27a). Ubiquitin degradated proteins particularly and selectively in cell and RPS27a is indispensable for translation. This multifunctional ribosomal protein is expressed at high levels in a wide variety of actively proliferating cells and tumor tissues and is a representative characteristic of various tumor cells. In our preliminary study of this protein in the silkworm,RPS27a also be found express highly in actively proliferating cells. The precise functional role of each ribosomal protein is largely unknown and many ribosomal proteins have extraribosomal functions apart from the particle. In this article, we review the recent research on the connection between tumor and this fusion protein, Ubiquitin-Proteasome Pathway and ribosomal protein. These research may indicate the origin and development of tumor, provide the basis for clinical diagnosis of cancer and the novel therapeutic targets for the treatment of malignant tumors.
Animals ; Biomarkers, Tumor ; Cloning, Molecular ; Humans ; Neoplasms ; genetics ; metabolism ; Recombinant Fusion Proteins ; metabolism ; Ribosomal Proteins ; biosynthesis ; genetics ; Ubiquitin ; biosynthesis ; genetics

OBJECTIVETo enhance the expression level of human papillomavirus (HPV) 16 L2E7 in Escherichia coli (E. coli), in aim of providing high-level expression of HPV16 L2E7 strain for pre-clinical high-throughout production.

METHODSThe whole L2E7 gene was optimized by software of Synthetic Gene Designer, reflecting E. coli codon usage. Two parts of codon-optimized gene were cloned into pET9a vector step by step. The positive clone, which was sequenced to be corrected, was transfected to BL21 (DE3+) via isopropyl-beta-D-thiogalactoside (IPTG) induction. They produced the HPV16 L2E7 fusion protein, which was further detected by SDS-PAGE and Western blot. The induction temperature, induction time, and IPTG concentration were also optimized by a series of experiments. Further purification modes of this protein were also explored.

RESULTSCodon-optimized HPV16 L2E7 was highly expressed in E. coli. The target protein accounted for nearly 60% of the total cell extract.

CONCLUSIONHigh-level expression of HPV16 L2E7 was successfully constructed.

Codon ; Escherichia coli ; genetics ; metabolism ; Human papillomavirus 16 ; metabolism ; Papillomavirus E7 Proteins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics

In order to detect the protein delivery mediated by the PTD (protein transduction domain) of TAT Protein, a expression vector, named pT7460-GFP, was constructed by insert the PTD DNA Sequence, followed by a GFP (green fluorescent protein) gene fused in-frame, into the pT7450 vector. The TAT-GFP fusion protein was expressed in the E. coli ER2566. Most of the fusion protein was presented in the inclusion body. The protein was purified by Ni2+ affinity chromatography under denature conditions, then by a Sepharose Q column to remove urea. The soluble denatured protein was added directly to medium containing the Myeloma Cell SP2/0. It came out that the fusion protein could be detected delivered into the cells under fluorescent microscope in a short time.
Gene Products, tat ; chemistry ; metabolism ; Green Fluorescent Proteins ; Humans ; Luminescent Proteins ; metabolism ; Microscopy, Fluorescence ; Multiple Myeloma ; metabolism ; Protein Transport ; Recombinant Fusion Proteins ; metabolism ; Tumor Cells, Cultured