Expression of chicken beta-actin in Saccharomyces cerevisiae

Actin interacts with a number of so-called actin-binding proteins which participate at various stages of the cell motility process such as regulation of filament formation, assembly and disassembly of filaments, force generation and depolymerization. Gene technology makes a precise mapping of the interacting surfaces on the actin molecules possible by studying specifically designed actin mutants expressed in a suitable organism. In addition, the production of engineered actin will become increasingly important when the three-dimensional structure of actin is determined. Chicken beta-actin can be produced in large quantities in Escherichia coli but such actin shows only a limited biological activity and thus seems to be of minor interest in future studies of structure-function relationships of this molecule. To circumvent the problem of a denatured bacterial protein, the yeast Saccharomyces cerevisiae was chosen as an alternative organism to express actin. This paper describes the expression, isolation and characterization of the yeast-produced chicken beta-actin. From a 12-liter culture of yeast cells, 500 micrograms of polymerizable beta-actin was isolated.     

A novel and highly efficient production system for recombinant adeno-associated virus vector

Recombinant adeno-associated virus(rAAV) has proven to be a promising gene delivery vector for human gene therapy. However, its application has been limited by difficulty in obtaining enough quantities of high-titer vector stocks. In this paper, a novel and highly efficient production system for rAAV is described. A recombinant herpes simplex virus type 1(rHSV-1) designated HSV1-rc/△UL2, which expressed adeno-associated virus type2(AAV-2) Rep and Cap proteins, was constructed previously. The data confirmed that its functions were to support rAAV replication and packaging, and the generated rAAV was infectious. Meanwhile, an rAAV proviral cell line designated BHK/SG2, which carried the green fluorescent protein(GFP) gene expression cassette, was established by transfecting BHK-21 cells with rAAV vector plasmid pSNAV-2-GFP. Infecting BHK/SG2 with HSV1-rc/△UL2 at an MOI of 0.1 resulted in the optimal yields of rAAV, reaching 250 transducing unit(TU) or 4.28×104 particles per cell. Therefore, compared with the conventional transfection method, the yield of rAAV using this "one proviral cell line, one helper virus" strategy was increased by two orders of magnitude. Large-scale production of rAAV can be easily achieved using this strategy and might meet the demands for clinical trials of rAAV-mediated gene therapy.     

A novel and highly efficient production system for recombinant adeno-associated virus vector

Recombinant adeno-associated virus (rAAV) has proven to be a promising gene delivery vector for human gene therapy. However, its application has been limited by difficulty in obtaining enough quantities of high-titer vector stocks. In this paper, a novel and highly efficient production system for rAAV is described. A recombinant herpes simplex virus type 1 (rHSV-1) designated HSV1-rc/AUL2, which expressed adeno-associated virus type2 (AAV-2) Rep and Cap proteins, was constructed previously. The data confirmed that its functions were to support rAAV replication and packaging, and the generated rAAV was infectious. Meanwhile, an rAAV proviral cell line designated BHK/SG2, which carried the green fluorescent protein (GFP) gene expression cassette, was established by transfecting BHK-21 cells with rAAV vector plasmid pSNAV-2-GFP. Infecting BHK/SG2 with HSV1-rc/AUL2 at an MOI of 0.1 resulted in the optimal yields of rAAV, reaching 250 transducing unit (TU) or 4.28×104 particles per cell. Therefore, compared     

Expression vectors based on the rac fusion promoter

The -35 region of the rrnB P2 promoter and the -10 region of the lacZpo promoter-operator were fused to form the strong and regulatable rac promoter. Vectors were constructed that allow the attachment of protein-coding sequences to the beta-galactosidase alpha-peptide (LacZ alpha) in any reading frame. By introducing a high-copy-number mutation, the synthesis of a LacZ alpha-chloramphenicol acetyltransferase fusion protein reached more than 60% of total cell protein in Escherichia coli.     

鸡白介素18成熟蛋白突变体在毕赤酵母中的高效表达及其生物活性检测

【目的】通过目的基因克隆,表达获得高产量具有生物活性的鸡白介素18成熟蛋白(maturechickeninterleukin-18,mChIL-18)。【方法】根据毕赤酵母密码子偏嗜性,通过引物设计来定点突变鸡白介素18基因,构建了重组质粒pPIC9K-mChIL-18。将线性化的重组阳性质粒电转到毕赤酵母GS115中,经筛选多拷贝阳性子后进行诱导表达。用MTT法和微量细胞病变抑制法检测其生物活性。【结果】筛选的多拷贝重组菌在诱导温度为28℃,培养液pH值为6.5,甲醇的诱导浓度为2%,诱导时间为120h时表达量最高,约为480mg/L。表达的mChIL-18蛋白能够刺激SPF鸡淋巴细胞大量增殖,用400μg/L的mChIL-18诱导淋巴细胞产生γ-干扰素(IFN-γ),其生物活性最高可达1.7×104U/mL,且能有效抑制水泡性口炎病毒(VSV)在鸡胚成纤维细胞(CEF)上的生长。【结论】毕赤酵母能够高效表达具有生物活性的鸡白介素18,有望作为免疫佐剂运用到工业化生产和兽医临床中。     

Regulation of the human beta-actin promoter by upstream and intron domains.

We have identified three regulatory domains of the complex human beta-actin gene promoter. They span a region of about 3000 bases, from not more than -2011 bases upstream of the mRNA cap site to within the 5' intron (832 bases long). A distal upstream domain contains at least one enhancer-like element. A proximal upstream domain, with a CArG [for CC(A + T rich)6GG] motif found in all known mammalian actin genes, seems to confer serum, but not growth factor, inducibility. The third domain is within the evolutionarily conserved 3' region of the first intron and contains a 13 base-pair sequence, identical to the upstream sequence with the CArG motif. This domain also contains sequences that are both serum and fibroblast growth factor inducible.     

WSSV ie1 promoter is more efficient than CMV promoter to express H5 hemagglutinin from influenza virus in baculovirus as a chicken vaccine

Background  

The worldwide outbreak of influenza A (H5N1) viruses among poultry species and humans highlighted the need to develop efficacious and safe vaccines based on efficient and scaleable production.     

High-cell-density fermentation for production of L-N-carbamoylase using an expression system based on the Escherichia coli rhaBAD promoter

A high-cell-density fed-batch fermentation for the production of heterologous proteins in Escherichia coli was developed using the positively regulated Escherichia coli rhaBAD promoter. The expression system was improved by reducing of the amount of expensive L-rhamnose necessary for induction of the rhamnose promoter and by increasing the vector stability. Consumption of the inducer L-rhamnose was inhibited by inactivation of L-rhamnulose kinase encoding gene rhaB of Escherichia coli W3110, responsible for the first irreversible step in rhamnose catabolism. Plasmid instability caused by multimerization of the expression vector in the recombination-proficient W3110 was prevented by insertion of the multimer resolution site cer from the ColE1 plasmid into the vector. Fermentation experiments with the optimized system resulted in the production of 100 g x L(-1) cell dry weight and 3.8 g x L(-1) of recombinant L-N-carbamoylase, an enzyme, which is needed for the production of enantiomeric pure amino acids in a two-step reaction from hydantoins.     

Expression of the murine interleukin-5 receptor on Xenopus laevis oocytes.

In this study we describe the use of Xenopus laevis oocytes for the detection of mRNA coding for a murine interleukin-5 (mI15) receptor. When injected with sucrose gradient fractionated polyA+ RNA derived from the murine 115-dependent pre B cell line B13, these oocytes could specifically bind 35S-methionine labeled mI15. A size of approximately 4000 nucleotides (25S) was estimated for the mRNA corresponding to the mIL5-binding activity. This binding was not blocked by a monoclonal antibody R52 specific for the MI15-receptor, suggesting that the oocytes express a different form of this receptor.     

New viral vector for efficient production of target proteins in plants

A new potato virus X (PVX)-based viral vector for superproduction of target proteins in plants has been constructed. The triple gene block and coat protein gene of PVX were substituted by green fluorescent protein. This reduced viral vector was delivered into plant cells by agroinjection (injection of Agrobacterium tumefaciens cells, carrying viral vector cDNA within T-DNA, into plant leaves), and this approach allowed to dramatically reduce the size of the vector genome. The novel vector can be used for production of different proteins including pharmaceuticals in plants.     

Chromosomal localization of chicken sequences homologous to the beta-actin gene by in situ hybridization

In situ DNA/chromosome hybridization techniques were used to localize the cytoplasmic beta-actin gene in the chicken. Hybridization of a beta-actin cDNA probe to metaphase chromosome spreads indicated that sequences complementary to this probe are located on the long arm of chromosome 2 (2q) and one of chromosomes 9 through 12.