Intein is a part of polypeptide in the premature protein with the capability of self-splicing, which is widely applied in protein purification, protein conjuction, cyclopeptide preparation, protein labeling and biosensor. In this review, we summarized the development of intein used in protein purification, discussed intein-mediated chromatographic and non-chromatographic purification systems, and summarized the researches in manipulating intein cleavage reaction. This work is to provide clues for improvement of intein-mediated protein purification.
Chromatography, Affinity ; Inteins ; Protein Splicing ; Proteins ; isolation & purification

To provide technical support for spider silk functional modification, we developed a simple and efficient functional platform via intein trans-splicing. Small ubiquitin-related modifier protein (SUMO) was fused to the recombinant spider silk protein (W2CT) by peptide bond via S0 split intein Ssp DnaB trans-splicing, resulting in a protein SUMOW2CT. However, incorporation of exogenous protein led to mechanical property defect and lower fiber yield, and also slowed down the fiber assembly velocity but no obvious differences in supercontraction and chemical resistance when compared with fibers from W2CT (W). SUMO protease digestion showed positive results on the fibers, indicating that the SUMO protein kept its native conformation and bioactive. Above all, this work provides a technical support for spider silk high simply and efficient functionalized modification.
Animals ; Inteins ; Protein Splicing ; Recombinant Proteins ; chemistry ; Silk ; chemistry ; Small Ubiquitin-Related Modifier Proteins ; chemistry ; Spiders ; Trans-Splicing

Alternative Splicing ; Fibronectins/genetics* ; Forensic Medicine ; Humans ; Protein Conformation ; Protein Isoforms/genetics* ; Wound Healing/physiology*

Alternative splicing (AS) is an important mechanism of producing transcriptome diversity and microarray techniques are being used increasingly to monitor the splice variants. There exist three types of microarrays interrogating AS events-junction, exon, and tiling arrays. Junction probes have the advantage of monitoring the splice site directly. Johnson et al., performed a genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays (Science 302:2141-2144, 2003), which monitored splicing at every known exon-exon junctions for more than 10,000 multi-exon human genes in 52 tissues and cell lines. Here, we describe an algorithm to deduce the relative concentration of isoforms from the junction array data. Non-negative Matrix Factorization (NMF) is applied to obtain the transcript structure inferred from the expression data. Then we choose the transcript models consistent with the ECgene model of alternative splicing which is based on mRNA and EST alignment. The probe-transcript matrix is constructed using the NMF-consistent ECgene transcripts, and the isoform abundance is deduced from the non-negative least squares (NNLS) fitting of experimental data. Our method can be easily extended to other types of microarrays with exon or junction probes.
Alternative Splicing ; Cell Line ; Exons* ; Humans ; Least-Squares Analysis ; Protein Isoforms ; RNA Precursors ; RNA, Messenger ; Transcriptome

CD44 is a hyaluronic acid receptor that exists as a standard 90-kd form (CD44H) as well as several CD44 variants isoforms are produced through alternative splicing. Alternatively spliced variants of the CD44 molecule have been found to be associated with invasive and metastatic potential of cancer cells and poor prognosis in several types of carcinoma. The purpose of the present study is to define the expression of CD44H and CD44v6 in ovarian tumors and to investigate whether the expression of these molecules is associated with adverse prognosis. We evaluated the expression of CD44 isoforms in 58 ovarian tumors by means of immunohistochemistry, and correlated between CD44 expression and the histologic types, tumor grade, peritoneal implants, pseudomyxoma peritonei and FIGO stage. While the CD44H was commonly expressed in ovarian tumors, the CD44v6 was expressed in a minor proportion of serous tumors in comparison with frequent expression of v6 isoform in mucinous tumors. The CD44H expression was significantly higher in stage I/II than in stage III. However, there was no correlation between the expression of CD44 and the presence of peritoneal implants or pseudomyxoma peritonei. These results suggest that CD44H could play an important role in the adhesive function in the lower stage of the ovarian tumor and reduced expression in the higher stage might be related to the metastasis and widespread invasion of ovarian carcinoma cells.
Adhesives ; Alternative Splicing ; Female ; Hyaluronic Acid ; Immunohistochemistry ; Mucins ; Neoplasm Metastasis ; Ovary ; Prognosis ; Protein Isoforms ; Pseudomyxoma Peritonei

High Mobility Group I(HMG-I) proteins are nuclear proteins that are required for induction of the human IFN-beta gene by virus and for the regulation of the tumor necrosis-beta factor and rRNA genes. Proteins I and Y result from alternative splicing of a single functional gene named HMGI(Y). In several studies, elevated expressions of the HMGI proteins (HMGI, HMGY, and HMGI-C) have been used as markers in thyroid cancer, but not in adenomas, goiters, and normal thyroid tissues and cells. Here, we try to demonstrate the elevated expression of the HMGI(Y) proteins in thyroid carcinomas by using semi-quantified RT-PCR (Reverse Transcription and Polymerase Chain Reaction). In cases of thyroid carcinomas 4 of 5(80%) were positive, in 10 cases of adenomas, goiters, and normal thyroid tissues, 1(10%) was positive. These results suggest that the semi-quantified RT-PCR is useful preoperative diagnostic tool for differentiating thyroid tumors.
Adenoma ; Alternative Splicing ; Genes, rRNA ; Goiter ; HMGA1a Protein ; Humans ; Nuclear Proteins ; Thyroid Gland* ; Thyroid Neoplasms*

PURPOSE: CD44 is a multifunctional adhesion molecule in cell-to-cell and cell-to-matrix interactions. This transmembrane glycoprotein exists in either standard or variant form, with the variation originating in alternative splicing. This study was designed to evaluate the role of CD44v6, one of the CD44 isoforms, in hepatocellular carcinoma and cholangiocarcinoma. MATERIALS AND METGODS: Immunohistochemical expression of CD44v6 was studied in 7 normal livers, 14 hepatocellular carcinomas and 16 cholangiocarcinomas, that were formalin fixed and paraffin embedded. RESULTS: CD44v6 was frequently expressed in the normal hepatocytes and hepatocellular carcinomas. Expression was not noted in the normal bile duct within the portal tract. CD44v6 was positively expressed in some of the proliferating bile ducts (43%) and cholangiocarcinomas (69%). CONCLUSION: CD44v6 expression may be more important in the stepwise carcinogenesis of the bile duct than in the normal hepatocyte, but further study is needed.
Alternative Splicing ; Bile Ducts ; Carcinogenesis ; Carcinoma, Hepatocellular* ; Cholangiocarcinoma* ; Formaldehyde ; Glycoproteins ; Hepatocytes ; Liver ; Paraffin ; Protein Isoforms

BACKGROUND: CD44 is the principal cell surface receptor for hyaluronate and exists as multiple isoforms generated by the alternative splicing of up to 10 variant exons. Although certain isofroms may play a role in tumor progression and metastasis formation, the precise function and expression of the variant isoforms are less clear. Since on normal eccrine glands CD44 standard isoform(CD44s) is expressed only in eccrine coil secretory cells, it can be considered as a possible marker of this type of differentiation. However little is known about the expression of CD44 variant isofroms(CD44v) in eccrine gland tumors. OBJECTIVE: The purpose of this study was to investigate the immunohistochemical expression of different CD44 isoforms(CD44s, CD44v4, CD44v6) in the eccrine gland tumors. METHODS: Formalin-fixed and paraffin-embedded tissues from 2 cases of eccrine hidrocystoma, 5 cases of syringoma, 2 cases of eccrine poroma, 2 cases of syringofibroadenoma, 2 cases of nodular hidradenoma were immunolabelled with monoclonal antibody directed CD44s, CD44v4, and CD44v6. RESULTS: Except for syringofibroadenoma, the most tumors cells with eccrine ductal differentiation showed negative staining for CD44s, and positive staining for CD44v4 and CD44v6. Syringofibroadenoma exhibited positive staining for CD44s and CD44v4, but negative staining for CD44v6. Eccrine poroma showed negative staining for CD44s, positive staining for CD44v4, and variable intensity of staining for CD44v6 in different areas of the tumors. In case of nodular hidradenoma, small tubular lumina and clear cells were positive for CD44s. CONCLUSION: Our results suggest that CD44 isoforms can not be a useful marker for an eccrine gland tumor with specific differentiation, but its characteristic pattern of distribution might reflect the variety of functional roles of CD44 isoforms in tumorigenesis of eccrine gland tumors.
Acrospiroma ; Alternative Splicing ; Carcinogenesis ; Eccrine Glands* ; Exons ; Hidrocystoma ; Negative Staining ; Neoplasm Metastasis ; Poroma ; Protein Isoforms* ; Syringoma

Coagulation factor VIII (fVIII) is a secretion protein and plays a crucial role in the coagulation cascade. Hemophilia A resulted from deficiency of fVIII is the most common X-linked recessive bleeding disorder. Gene therapy is recognized as an attractive strategy for the eventual cure of this disease. However, the gene therapy is hampered by the big size of fVIII gene when using the most promising gene vectors, adeno-associated virus (AAV) vectors. In this study we explored the intein-mediated protein trans-splicing to deliver a Phe(309)-->Ser mutant full-length fVIII (F309SfVIII) gene by using a dual-vector system. An intein is a protein sequence embedded within a precursor protein and can excise itself through protein splicing. The F309SfVIII is proven to be beneficial to its secretion. The F309SfVIII gene was broken into heavy and light chains before Ser(1239) in B domain and fused with the coding sequences of Ssp DnaB intein respectively to construct a pair of plasmid vectors by inserting them into the pcDNA3.1 vectors. Forty-eight hours after co- or separate transfection of 293 cells, the co-transfected cell lysate showed an obvious ligated F309SfVIII protein band by Western blot with a polyclonal antibody against fVIII. The amounts of secreted F309SfVIII protein in culture supernatants and their bioactivities were (71+/-9) ng/mL and (0.38+/-0.09) IU/mL determined by ELISA and Coatest assay respectively. The supernatant from combined cells with separate transfections also displayed lower levels of F309SfVIII antigen and fVIII activity [(25+/-6) ng/mL and (0.12+/-0.05) IU/mL], indicating the F309SfVIII could be formed by splicing both before and after secretion. The content of F309SfVIII heavy chain protein from co-transfected cell supernatant was higher than that of intein-fused heavy chain transfection alone [(135+/-10) ng/mL vs (37+/-7) ng/mL, P<0.01)]. These data demonstrated that intein could be used as a technical strategy in a dual-vector system delivering F309SfVIII gene with improved secretion of fVIII providing an alternative approach to circumvent the packaging limitation of AAV for F309SfVIII gene transfer, which encourages our continuing study in hemophilia A gene therapy in vivo.
Cell Line ; Dependovirus ; Factor VIII ; metabolism ; Genetic Vectors ; Humans ; Inteins ; Protein Splicing

Hypertonicity imposes a great deal of stress to cells since it causes rise in cellular ionic strength, which can be reduced by the accumulation of compatible osmolytes. TonEBP plays a central role in the cellular accumulation of compatible osmolytes via transcriptional stimulation of membrane transporters and aldose reductase. Alternatively spliced forms of TonEBP mRNA have previously been reported and two of them showed different transcriptional activity. In the present study, isoform-specific antibodies were produced to confirm the translation of the spliced mRNA to protein. TonEBP was immunoprecipitated by using anti-TonEBP antibody and then immunoblotted using anti-TonEBP or isoform specific antibodies to find out the expression profile of TonEBP isoforms in basal or stimulated condition. From these results, we conclude that all TonEBP isoforms are expressed in mammalian cells and their expression patterns are not same in every cells.
Aldehyde Reductase ; Alternative Splicing ; Antibodies ; Membrane Transport Proteins ; Osmolar Concentration ; Protein Isoforms* ; RNA, Messenger