Reduced nitrogen fertilization from pre-flowering to pre-veraison alters phenolic profiles of Vitis vinifera L. Cv. Cabernet Gernischt wine of Yantai, China.

Nitrogen (N) fertilization is important for grape growth and wine quality. Unreasonable N fertilizer application affects wine growth and has a negative impact on wine quality. Therefore, it is essential to address the mismatch between N application and wine composition. To regulate vine growth and improve grape and wine quality, Cabernet Gernischt (Vitis vinifera L.) grapevines were subjected to lower levels of N, compared to normal N supply treatments, during the grape growing seasons of 2019 and 2020 in the wine region of Yantai, China. The effects of reduced N application from pre-boom to pre-veraison on vine growth, yield and composition of grapes, and dry red wine anthocyanin and non-anthocyanin phenolic compound content were studied. We found that reduced N application significantly decreased dormant shoot fresh mass and yield. However, the effect of N application on fruit ripening depended on the season. Nitrogen-reduction treatment significantly improved wine phenolic parameters, including total phenolics, tannins, and anthocyanins, and enhanced most of the individual anthocyanins and some non-anthocyanin phenolics, especially stilbenes, including piceatannol, trans-resveratrol, and polydatin, regardless of the season. Overall, our findings highlight the importance of reducing N application during the grape growing season in order to modify the wine phenolic profiles.

Modification of wine phenolic profiles by gibberellic acid application in 'Cabernet Gernischt' grapevines before anthesis.

BACKGROUND: Vitis vinifera L. 'Cabernet Gernischt' grapes from the Yantai wine region of China usually form dense clusters and contain low phenolic content. We applied five concentrations (ranged from 5 to 25 mg L-1 ) of gibberellic acid (GA3 ) to 'Cabernet Gernischt' before anthesis to decrease cluster compactness in two consecutive vintages. Yield indices, grape maturity, and wine phenolic compounds were determined. RESULTS: GA3 application significantly reduced cluster compactness, bunch weight, and yield per vine, but it did not significantly improve fruit ripening. The levels of total phenolics, total tannins, and total anthocyanins in wine were enhanced by GA3 application, with 10 and 15 mg L-1 GA3 treatments consistently producing a significant increase in the concentrations of mavidin, cyanidin, and their derivatives. Specifically, trans-resveratrol was consistently significantly increased by 15 mg L-1 GA3 application. Principal component analysis of phenolic compounds demonstrated the differences among wines produced from GA3 treatment groups and the control. CONCLUSION: Overall, wine phenolic profiles could be significantly modified by application of low concentrations of GA3 before anthesis. Application of high levels of GA3 is not recommended due to significant yield decrease. © 2022 Society of Chemical Industry.

Co-winemaking with Vitis amurensis Rupr. "Beibinghong" enhances the quality of Vitis vinifera L. cv. Cabernet Gernischt wine.

In some wine regions of China, Cabernet Gernischt (CG; Vitis vinifera L.) grape berries usually exhibit low pigment content and titratable acidity, and low sensory quality of the resulting wine. The aim of this study was to evaluate co-winemaking of CG wines using the red grape cultivar Beibinghong (BBH; Vitis amurensis Rupr.) at different proportions in terms of alcohols, phenolic compounds, and sensory properties of the wines. The results showed that the co-winemaking wines contained a similar content of higher alcohols, whereas the methanol content increased with an increase in BBH proportion, although this was still corresponded with the national standard. Significantly higher levels of titratable acidity were observed in co-winemaking wines at the ratio of 6:4 and 5:5, compared with monocultivar CG wines. All co-winemaking wines, except CG:BBH (9:1) wine, showed significantly higher levels of total anthocyanins, total phenolics, total tannins, and total flavan-3-ols. Further, individual phenolics, primarily diglucoside anthocyanins and non-anthocyanins (trans-ferulic acid, myricetin, viniferin, trans-caffeic acid, 3,4-dihydroxybenzoic acid), as important contributors to wine color intensity, permitted the differentiation of the wines via principal component analysis. In most cases, co-winemaking wines exhibited higher scores of the 10 sensory attributes on color, aroma, mouthfeel, and overall quality compared with monocultivar wines. Co-winemaking CG wines with BBH at 7:3 ratio demonstrated the highest scores of color intensity, aroma intensity, aroma quality, and overall quality. The results indicate that co-winemaking with V. amurensis grape variety may be useful to enhance V. vinifera wine quality by modifying wine composition. PRACTICAL APPLICATION: Cabernet Gernischt (CG) is the predominant grape cultivar used to prepare premium-quality wine in China; however, in some wine regions, CG wines have low levels of pigment and titratable acidity, and low sensory quality. Co-winemaking with another native grape cultivar, Beibinghong (BBH), which is characterized by a higher content of anthocyanins and acidity, provided sufficient experimental evidence of adjustments in the Vitis vinifera wine composition leading to improved wine sensory quality.

Pip serves as an intermediate in RpoS-modulated phz2 expression and pyocyanin production in Pseudomonas aeruginosa.

Pyocyanin, a main virulence factor that is produced by Pseudomonas aeruginosa, plays an important role in pathogen-host interaction during infection. Two copies of phenazine-biosynthetic operons on genome, phz1 (phzA1B1C1D1E1F1G1) and phz2 (phzA2B2C2D2E2F2G2), contribute to phenazine biosynthesis. In our previous study, we found that RpoS positively regulates expression of the phz2 operon and pyocyanin biosynthesis in P. aeruginosa PAO1. In this work, when a TetR-family regulator gene, pip, was knocked out, we found that pyocyanin production was dramatically reduced, indicating that Pip positively regulates pyocyanin biosynthesis. With further phenazines quantification and ß-galactosidase assay, we confirmed that Pip positively regulates phz2 expression, but does not regulate phz1 expression. In addition, while the rpoS gene was deleted, expression of pip was down-regulated. Expression of rpoS in the wild-type PAO1 strain, however, was similar to that in the Pip-deficient mutant PAΔpip, suggesting that expression of pip could positively be regulated by RpoS, whereas rpoS could not be regulated by Pip. Taken together, we drew a conclusion that Pip might serve as an intermediate in RpoS-modulated expression of the phz2 operon and pyocyanin biosynthesis in P. aeruginosa.

Enhanced expression of GCRV VP6 in CIK cells by relative sequence optimization.

Efficient expression of target protein is one of strategies for gene therapy or vaccine design. Many studies showed that codon optimization could enhance the expression of target proteins. In this paper, a target sequence of about 1.26 kb encoding the major capsid protein VP6 of grass carp reovirus (GCRV) and an optimized counterpart were synthesized and inserted into vectors for expressing VP6. The final constructs (named as pcDV6G and pcDV6YG) were transfected in Ctenopharyngodon idellus kidney (CIK) cells. The fluorescence analysis and the Western blot results showed that the gene fragment was transfected and expressed in CIK cells successfully. Although the qRT-PCR results showed no difference at the messenger RNA (mRNA) levels between the different versions of vp6 in the indicated stages, the enzyme-linked immunosorbent assay (ELISA) results showed that the protein level of VP6 expressed by pcDV6YG was higher than that by pcDV6G in the indicated hours. Taken together, these results suggest that the enhanced expression of GCRV VP6 in CIK cells by relative sequence optimization may be a good choice for making DNA vaccine against GCRV.

Inter-chain disulfide bond improved protein trans-splicing increases plasma coagulation activity in C57BL/6 mice following portal vein FVIII gene delivery by dual vectors.

Protein trans-splicing based dual-vector factor VIII (FVIII) gene delivery is adversely affected by less efficiency of protein splicing. We sought to increase the amount of spliced FVIII protein and plasma coagulation activity in dual-vector FVIII transgene in mice by means of strengthening the interaction of inteins, protein splicing elements, thereby facilitating protein trans-splicing. Dual-vector delivery of the FVIII gene in cultured cells showed that replacement of Met226 in the heavy chain and Asp1828 in the light chain with Cys residues could facilitate inter-chain disulfide linking and improve protein trans-splicing, increasing the levels of spliced FVIII protein. In this study, C57BL/6 mice were coadministered dual vectors of intein-fused human FVIII heavy chain and light chain with Cys mutations via portal vein injection into the liver. Forty-eight hours post-injection, plasma was collected and analyzed for FVIII antigen concentration and coagulation activity. These mice showed increased circulating FVIII heavy chain polypeptide (442 ± 151 ng m(L-1) vs. 305 ± 103 ng mL(-1)) and coagulation activity (1.46 ± 0.37 IU m(-1) vs. 0.85 ± 0.23 IU mL(-1)) compared with control mice co-administered dual vectors expressing the heavy and light chains of wild-type FVIII. Moreover, coagulation activity was similar to that of mice receiving a single vector expressing FVIII (1.79 ± 0.59 IU mL(-1)). These findings indicate that improving protein trans-splicing by inter-chain disulfide bonding is a promising approach for increasing the efficacy of dual-vector based FVIII gene transfer.

[Trans-splicing of Cys mutated coagulation factor VIII].

To investigate the improving effect of inter-chain disulfide formation on protein trans-splicing, we introduce a Cys point mutation at Tyr(664) in heavy chain and at Thr(1826) in light chain of B-domain-deleted FVIII (BDD-FVIII). By co-transfection of COS-7 cell with the two Cys mutated chain genes, the intracellular protein splicing, inter-chain disulfide formation, secreted BDD-FVIII and bioactivity in culture supernatant were observed. The data showed that a strengthened spliced BDD-FVIII with an inter-chain disulfide detected by Western blotting and an elevated secretion of spliced BDD-FVIII (128 +/- 24 ng mL(-1)) compared to control (89 +/- 15 ng mL(-1)), assayed by a sandwich ELISA. A Coatest was performed to assay the secretion of bioactivity in culture supernatant and shown a much higher value (0.94 +/- 0.08 u mL(-1)) compared to that of control (0.62 +/- 0.15 u mL(-1)). It suggests that inter-chain disulfide formation could improve protein trans-splicing based dual-vector delivery of BDD-FVIII gene providing experimental evidence for ongoing in vivo study.

Enhanced plasma factor VIII activity in mice via cysteine mutation using dual vectors.

Hemophilia A is caused by a genetic mutation in coagulation factor VIII (FVIII) gene and gene therapy is considered to be a promising strategy for its treatment. We recently demonstrated that co-delivery of two vectors expressing M662C mutated heavy and D1828C mutated light chain genes of B-domain-deleted coagulation factor VIII (BDD-FVIII) leads to inter-chain disulfide cross-linking and improved heavy chain secretion in vitro. In this study, co-injection of both M662C and D1828C mutated BDD-FVIII gene expression vectors into mice resulted in increased heavy chain secretion and coagulation activity in plasma in vivo. Approximately (239 ± 56) ng mL(-1) above endogenous levels of transgenic FVIII heavy chain was found in mouse plasma using a chain-specific ELISA. For FVIII coagulation activity, approximately (1.09 ± 0.25) IU mL(-1) above endogenous levels were detected in co-injected transgenic mouse plasma using a chromogenic assay. These data demonstrate that inter-chain disulfide bonds likely increase heavy chain secretion and coagulation activity in the plasma of transgenic mice with an improved efficacy of a dual-vector delivery of BDD-FVIII gene. These findings support our ongoing efforts to develop a gene therapy for hemophilia A treatment using dual-AAV vectors.

[Leucine zippers improves protein splicing-mediated coagulation factor VIII gene delivery by dual-vector system].

In our recent study by exploring an intein-based dual-vector to deliver a B-domain-deleted FVIII (BDD-FVIII) gene, it showed that covalently ligated intact BDD-FVIII molecules with a specific coagulant activity could be produced from expressed heavy and light chains by protein trans-splicing. Here, we assessed the hypothesis that the efficiency of trans-splicing may be increased by adding to the intein sequences a pair of leucine zippers that are known to bring about specific and strong protein binding. The intein-fused heavy and light chain genes were co-transferred into cultured COS-7 cells using a dual-vector system. After transient expression, the intracellular BDD-FVIII splicing was observed and the spliced BDD-FVIII and bioactivity secreted to culture media were quantitatively analyzed. An enhanced splicing of BDD-FVIII with decreased protein precursors from gene co-transfected cells was observed by Western blotting. The amount of spliced BDD-FVIII and bioactivity secreted to the culture media were 106 +/- 12 ng x mL(-1) and 0.89 +/- 0.11 U x mL(-1) analyzed by ELISA and Coatest method respectively, which was greater than leucine zipper free intein-fused heavy and light chain genes co-transfected cells (72 +/- 10 ng x mL(-1) and 0.62 +/- 0.07 U x mL(-1)). The activity of cellular mechanism-independent protein splicing was also improved, as showed by the increasing of spliced BDD-FVIII and bioactivity in culture media from combined cells separately transfected with heavy and light chain genes which was 36 +/- 11 ng x mL(-1) and 0.28 +/- 0.09 U x mL(-1). It demonstrated that the leucine zippers could be used to increase the efficiency of protein trans-splicing to improve the efficacy of a dual-vector mediated BDD-FVIII gene delivery by strengthening the interaction between the two intein-pieces fused to heavy and light chains. It provided evidence for further study in animal model using a dual-adeno-associated virus vector to deliver FVIII gene in vivo.

[Enhancing effect of deoxynivalenol-mediated GRP78 down-regulation on heavy chain secretion and bioactivity of two-chain FVIII gene co-transfected cells].

Although two chain transfering separately could be used to overcome the volume limitation of adeno-associated virus vectors (AAV) in coagulation factor VIII (FVIII) gene delivery, it leads to chain imbalance for inefficient heavy chain secretion. In this study we aimed to improve the efficacy of two chain strategy in FVIII gene delivery through the degradation of glucose-regulated protein 78 (GRP78) known as a protein chaperone in endoplasmic reticulum (ER) by deoxynivalenol (DON) to decrease GRP78-bound FVIII heavy chain. By treating the two-chain gene transduced 293 cells with DON, the heavy chain (HC) secretion and FVIII bioactivity were observed. Data showed that 293 cells after three hours post-treatment with DON at a concentration of 500 ng mL(-1) resulted in obvious decrease the level of GRP78 but no effect on the cell proliferation. The HC secreted from DON-treated cells transfected with HC gene alone was 59 +/- 11 ng mL(-1), higher than that secreted by control cells (15 +/- 4 ng mL(-1)), and the HC secretion was further increasing to 146 +/- 34 ng mL(-1) in light chain (LC) gene co-transfected cells with an activity measured up to 0.66 +/- 0.15 U mL(-1), also greater than control cells (76 +/- 17 ng mL(-1) and 0.35 +/- 0.09 U mL(-1)). Taken together, these data suggest that DON-mediated GRP78 down-regulation could improve the efficacy of two-chain FVIII gene transfering by facilitating HC secretion, providing an experimental basis for in vivo dual-AAV application in FVIII gene delivery.

[Glycosylation modifications improve secretion and activity of intein spliced coagulation factor VIII].

OBJECTIVE: to investigate the effect of glycosylation modification on secretion of intein spliced B-domain-deleted FVIII (BDD-FVIII). METHODS: a total of 226 amino acid residues of FVIII B domain with six potential asparagines-linked glycosylation sites (N6) were incorporated into heavy chain of BDD-FVIII. By dual-vector co-transfer of heavy and light chain genes with fused intein (N6HCIntN and IntCLC) into cultured 293 cells, the amounts of spliced BDD-FVIII protein and coagulation activity in culture supernatant were observed by ELISA and Coatest method respectively. RESULTS: the amounts of spliced BDD-FVIII protein and activity were up to (123 ± 18) ng/ml and (0.94 ± 0.11) U/ml in supernatant from cells co-transfected with N6HCIntN and IntCLC genes. And they were higher than those of cells co-transfected with intein-fused heavy (HCIntN) and light chain (IntCLC) genes [(86 ± 12) ng/ml and (0.65 ± 0.07) U/ml, both P < 0.05]. Spliced BDD-FVIII protein and activity could also be detected in the supernatant from mixed cells individually transfected with N6HCIntN and IntCLC genes [(18 ± 6) ng/ml and (0.15 ± 0.05) U/ml]. CONCLUSION: it demonstrated that the glycosylation modified heavy chain can improve the secretion of intein spliced BDD-FVIII and the protein splicing can occur independent of cellular mechanism.

[vWF improves secretion and activity of intein spliced BDD-FVIII].

As synthesized by vascular endothelial cells and megakaryocytes, the von Willebrand factor (vWF) plays an important hemostatic role in the binding to and stabilizing blood coagulation factor VIII (FVIII) and preventing its enzymatic degradation. Our recent work demonstrated intein can efficiently ligate BDD-FVIII (B-domaim deleted FVIII) posttranslationally by protein trans-splicing after transfer of split BDD-FVIII gene by a dual-vector system. In this study we investigated the effect of vWF on secretion and activity of intein-ligated BDD-FVIII. We observed the levels of full-length BDD-FVIII antigen secreted into culture supernatant by ELISA and their activity by Coatest assay after transfection of cultured 293 cells with intein-fused BDD-FVIII heavy- and light-chain genes simultaneously with the vWF gene co-transfected. The data showed that the amount of full-length BDD-FVIII protein and their bioactivity in vWF gene co-transfected cell supernatant were 235 +/- 21 ng x mL(-1) and 1.98 +/- 0.2 u x mL(-1), respectively, greater than that of non-vWF co-transfected cell (110 +/- 18) ng x mL(-1) and 1.10 +/- 0.15 u x nL(-1)) or just BDD-FVIII gene transfected control cell (131 +/- 25 ng x mL(-1) and 1.22 +/- 0.18 u x mL(-1)) indicating the benefit of vWF gene co-transfection in the secretion and activity of intein-spliced BDD-FVIII protein. It provided evidence that vWF gene co-transfer may be useful to improve efficacy of gene therapy for hemophilia A in protein splicing-based split FVIII gene transfer.

[Enhancing effect of porcine coagulation factor VIII A1 and A3 domains on secretion of post-translationally spliced human/porcine hybrid coagulation factor VIII].

Low levels of coagulation factor VIII (fVIII) protein expression caused by its inefficient secretion and the over-sized fVIII gene affect the transgene-based gene therapy for hemophilia A adversely. Our previous study demonstrated that intein-mediated protein trans-splicing for delivery of the fVIII gene with a dual-vector system could improve secretion of post-translationally spliced fVIII by light chain in cis. In this study, a human/porcine hybrid fVIII (HP-fVIII) containing replaced A1 and A3 domains of porcine fVIII was investigated for secretion and activity of the spliced HP-fVIII after intein-based dual-vector delivery of the HP-fVIII gene. A pair of expression plasmids comprising intein-fused HP-fVIII heavy and light chains were constructed and transiently co-transfected into COS-7 cells. The spliced HP-fVIII and bio-activity in culture media were quantitatively analyzed by ELISA and Coatest method respectively. The intracellular splicing of HP-fVIII was detected by Western blotting. The results showed that in the culture supernatant of cells co-transfected with HP-fVIII, the amount and activity of spliced HP-fVIII were significantly higher than those of spliced hfVIII secreted from the cells co-transfected with human fVIII [(184+/-34 ng/mL) vs (48+/-12) ng/mL, P<0.01; (1.18+/-0.22) IU/mL vs (0.31+/-0.10) IU/mL, P<0.01], demonstrating the dramatically enhancing effect of porcine A1 and A3 domains on the secretion of intein-spliced HP-fVIII. The spliced HP-fVIII protein and its activity were also detected in the supernatant from combined cells separately transfected with intein-fused HP-fVIII heavy and light chain genes, indicating that the intein-mediated HP-fVIII splicing was independent of cellular mechanism and could occur outside the cell after the secretion of precursor proteins. Additionally, an intracellularly spliced HP-fVIII band was found with a molecular weight similar to human fVIII protein, confirming the HP-fVIII splicing. These results provided experimental basis for ongoing study using intein-based dual adeno-associated virus (AAV) vector to transfer HP-fVIII gene in animal models.

Protein trans-splicing based dual-vector delivery of the coagulation factor VIII gene.

A dual-vector system was explored for the delivery of the coagulation factor VIII gene, using intein-mediated protein trans-splicing as a means to produce intact functional factor VIII post-translationally. A pair of eukaryotic expression vectors, expressing Ssp DnaB intein-fused heavy and light chain genes of B-domain deleted factor VIII (BDD-FVIII), was constructed. With transient co-transfection of the two vectors into 293 and COS-7 cells, the culture supernatants contained (137+/-23) and (109+/-22) ng mL(-1) spliced BDD-FVIII antigen with an activity of (1.05+/-0.16) and (0.79+/-0.23) IU mL(-1) for 293 and COS-7 cells, respectively. The spliced BDD-FVIII was also detected in supernatants from a mixture of cells transfected with inteinfused heavy and light chain genes. The spliced BDD-FVIII protein bands from cell lysates were visualized by Western blotting. The data demonstrated that intein could be used to transfer the split factor VIII gene and provided valuable information on factor VIII gene delivery by dual-adeno-associated virus in hemophilia A gene therapy.

[Glycosylation and L303e/F309S mutations improve intein-mediated splicing of the split coagulation factor VIII].

We recently demonstrated that an intein-mediated protein splicing can be used to transfer B-domain-deleted FVIII (BDD-FVIII) gene by a dual-vector. In this study, we observed the effect of a variant heavy chain with six potential glycosylation sites of B domain and L303E/F309S mutations in its A1 domain, which were proven to be beneficial for FVIII secretion, on secretion of spliced BDD-FVIII. By transient co-transfection of cultured 293 cells with intein-fused variant heavy chain (DMN6HCIntN) and light chain (IntCLC) genes, the culture supernatant was analyzed quantitatively by ELISA for secreted spliced BDD-FVIII antigen and by a chromogenic assay for bioactivity. The data showed that the amount of spliced BDD-FVIII protein and coagulation activity in culture supernatant from DMN6HCIntN plus IntCLC co-transfected cells were up to (149 +/- 23) ng x mL(-1) and (1.12 +/- 0.14) u x mL(-1) respectively greater than that of intein-fused wild type heavy (HCIntN) and light chain (IntCLC) co-transfected cells [(99 +/- 14) ng x mL(-1) and (0.77 +/- 0.13) u x mL(-1)] indicating that the variant heavy chain is able to improve the secretion of spliced BDD-FVIII and activity. A cellular mechanism-independent BDD-FVIII splicing was also observed. It provided evidence for ongoing animal experiment using intein-mediated dual-AAV vector technology for delivery of the BDD-FVIII genes.

[Protein trans-spliced chimeric human/porcine BDD-FVIII with augmented secretion].

This study is to construct a chimeric human/porcine BDD-FVIII (BDD-hpFVIII) containing the substituted porcine A1 and A3 domains which proved to have a pro-secretory function. By exploring Ssp DnaB intein's protein trans-splicing a dual-vector was adopted to co-transfer the chimeric BDD-hpFVIII gene into cultured COS-7 cell to observe the intracellular BDD-hpFVIII splicing by Western blotting and secretion of spliced chimeric BDD-hp FVIII protein and bio-activity using ELISA and Coatest assay, respectively. The dada showed that an obvious protein band of spliced BDD-hpFVIII can be seen, and the amount of spliced BDD-hpFVIII protein and bio-activity in the supernatant were up to (340 +/- 64) ng x mL(-1) and (2.52 +/- 0.32) u x mL(-1) secreted by co-transfected cells which were significantly higher than that of dual-vector-mediated human BDD-FVIII gene co-transfection cells [(93 +/- 22) ng x mL(-1), (0.72 +/- 0.13) u x mL(-1)]. Furthermore, a spliced BDD-hpFVIII protein and activity can be detected in supernatant from combined cells separately transfected with intein-fused BDD-hpFVIII heavy and light chain genes indicating that intein-mediated BDD-hpFVIII splicing occurs independently of cellular mechanism. It provided evidence for enhancing FVIII secretion in the research of animal models using intein-based dual vector for the delivery of the BDD-hpFVIII gene.

[Enhancing effect of AR-3 on protein splicing-based eukaryotic cell gene transfer of full-length coagulation factor VIII].

OBJECTIVE: To study the effect of an acidic region-3 (AR-3), capable of improving the secretion of heavy chain of coagulation factor VIII (fVIII), on the secretion of protein spicing ligated full-length fVIII. METHODS: A pair of vectors was used to deliver intein fused heavy and light chain genes of a full-length fVIII gene with an additional AR-3 incorporated on the end of heavy chain into cultured 293 cells. The intracellular protein splicing was observed by Western blot. And the secretion of spliced fVIII and activity in culture supernatant were quantified by enzyme-linked immunosorbent assay (ELISA) and Coatest assay respectively. RESULTS: A noticeable spliced fVIII protein band was observed from the gene co-transfected cells. The culture supernatant displayed a spliced fVIII of (112 ± 18) ng/ml with an activity of (0.76 ± 0.13) U/ml greater than that of cells co-transfected with AR-3-free heavy chain and light chain genes [(64 ± 11) ng/ml and (0.37 ± 0.05) U/ml]. And a spliced fVIII of (27 ± 7) ng/ml with an activity of (0.16 ± 0.05) U/ml was detected in the culture supernatant from combined cells separately transfected with AR-3-fused heavy chain gene and light gene. CONCLUSION: AR-3 can enhance the fVIII gene transfer by improving the secretion of intein spliced full-length fVIII.

[Post-translational ligation of split CFTR severed before TMD2 and its chloride channel function].

Mutations of cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to cystic fibrosis, an autosomal recessive genetic disorder affecting a number of organs including the lung airways, pancreas and sweat glands. In order to investigate the post-translational ligation of CFTR with reconstructed functional chloride ion channel and the split Ssp DnaB intein-mediated protein trans-splicing was explored to co-deliver CFTR gene into eukaryotic cells with two vectors. The human CFTR cDNA was split after Glu838 codon before the second transmembrane dome (TMD2) into two halves of N- and C-parts and fused with the coding sequences of split Ssp DnaB intein. Pair of eukaryotic expression vectors pEGFP-NInt and pEYFP-IntC were constructed by inserting them into the vectors pEGFP-N1 and pEYFP-N1 respectively. The transient expression was carried out for observing the ligation of CFTR by Western blotting and recording the chloride current by patch clamps when cotransfection of the pair of vectors into baby hamster kidney (BHK) cells. The results showed that an obvious protein band proven to be ligated intact CFTR can be seen and a higher chloride current and activity of chloride channel were recorded after cotransfection. These data demonstrated that split Ssp DnaB intein could be used as a strategy in delivering CFTR gene by two vectors providing evidence for application of dual adeno-associated virus (AAV) vectors to overcome the limitation of packaging size in cystic fibrosis gene therapy.

[Post-translational ligation and function of dual-vector transferred split CFTR gene].

The mutation of cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to an autosomal recessive genetic disorder cystic fibrosis (CF). The gene therapy for CF using adeno-associated virus (AAV) vectors delivering CFTR gene is restricted by the contents limitation of AAV vectors. In this study the split CFTR genes severed at its regulatory domain were delivered by a dual-vector system with an intein-mediated protein trans-splicing as a technique to investigate the post-translational ligation of CFTR half proteins and its function as a chloride ion channel. A pair of eukaryotic expression vectors was constructed by breaking the human CFTR cDNA before Ser712 codon and fusing with Ssp DnaB intein coding sequences. After co-transfection into baby hamster kidney (BHK) cells followed by transient expression, patch clamps were carried out to record the chloride current of whole-cell and the activity of a single channel, and the ligation of two halves of CFTR was observed by Western blotting. The results showed that the intein-fused half genes co-transfected cells displayed a high whole cell chloride current and activity of a single channel indicating the functional recovery of chloride channel, and an intact CFTR protein band was figured out by CFTR-specific antibodies indicating that intein can efficiently ligate the separately expressed half CFTR proteins. The data demonstrated that protein splicing strategy could be used as a strategy in delivering CFTR gene by two vectors, encouraging our ongoing research program on dual AAV vector system based gene transfer in gene therapy for cystic fibrosis.

[Intein-mediated F309SfVIII ligation with enhanced secretion of its heavy chain.].

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.