Vectors expression

Gene Expression Systems. One of the potentials of genetic engineering of microbes is production of large amounts of recombinant proteias (12,13). This is not a trivial task. Each proteia is unique and the stabiUty of the proteia varies depending on the host. Thus it is not feasible to have a single omnipotent microbial host for the production of all recombinant proteias. Rather, several microbial hosts have to be studied. Expression vectors have to be tailored to the microbe of choice. 

J. Vlak and R. Keus, "Baculovims Expression Vector System for Production of Viral Vacciues," iu A. Mizrahi, ed.. Viral Vaccines, Wiley-Liss, New York, 1990. 

LDH-x is one of the best characterized antigens and its amino acid sequence is known. A synthetic peptide based on a portion of the molecule has been shown to reduce fertUity in laboratory animals. The nucleotide sequence coding for human LDH-x has been defined and engineered into an expression vector system (121). 

Smith, G.P. Filamentous fusion phage novel expression vectors that display cloned antigens on the virion surface. Science 228 1315-1317, 1985. 

Expression vectors are engineered so that any cloned insert can be transcribed into RNA, and, in many instances, even translated into protein. cDNA expression libraries can be constructed in specially designed vectors derived from either plasmids or bacteriophage A. Proteins encoded by the various cDNA clones within such expression libraries can be synthesized in the host cells, and if suitable assays are available to identify a particular protein, its corresponding cDNA clone can be identified and isolated. Expression vectors designed for RNA expression or protein expression, or both, are available. 

A vector for in vitro expression of DNA inserts as RNA transcripts can be constructed by putting a highly efficient promoter adjacent to a versatile cloning site. Figure 13.15 depicts such an expression vector. Linearized recombinant vector DNA is transcribed in vitro using SPG RNA polymerase. Large amounts of RNA product can be obtained in this manner if radioactive ribonucleotides are used as substrates, labeled RNA molecules useful as probes are made. 

FIGURE 13.15 Expression vectors carrying the promoter recognized by the RNA polymerase of bacteriophage SPG are useful for making RNA transcripts in vitro. SPG RNA polymerase works efficiently in vitro and recognizes its specific promoter with high specificity. 

These vectors typically have a polylinker adjacent to the SPG promoter. Successive rounds of transcription initiated by SPG RNA polymerase at its promoter lead to the production of multiple RNA copies of any DNA inserted at the polylinker. Before transcription is initiated, the circular expression vector is linearized by a single cleavage at or near the end of the insert so that transcription terminates at a fixed point. 

FIGURE 13.17 A protein expression vector contains the hybrid promoter derived from fusion of the lac and trp promoters. Expression from pi A more than 10 times greater than expression from either the lac or trp promoter alone. Isopropyl-/3-D-thiogalacto-side, or IPTG, induces expression from pi as well as lac. 

The field of DNA vaccination started when eukaryotic expression vectors were injected into the muscle of laboratory animals [2]. The authors observed protein expression for more than 2 months after injection and noted that no special delivery system was required to obtain this expression. Subsequently, it was demonstrated that antibodies can be induced simply by injecting plasmid DNA into the muscle of mice [3]. Subsequent studies found that the injection of expression plasmids also leads to the induction of a cytotoxic T-cell response. After injection, the DNA enters cells of the vaccinated host and the encoded gene becomes expressed. This eventually leads to the induction of a cellular cytotoxic T-cell, T-helper, and/or humoral (antibody) immune response. 

There are various protocols of administering eukaryotic expression vectors aiming to deliver (i.e. transfect) the DNA into the cytoplasm of the host cells (see Figure 2). The DNA is subsequently imported into the nucleus of the transfected cells allowing expression of the... 

The eukaryotic expression cassette is the part of an expression vector that enables production of a protein in a eukaryotic cell. The cassette consists of a eukaryotic promoter for mRNA transcription, the gene and an mRNA termination and processing signal (Poly-A signal). 

PelZ is a hydrophilic protein of 420 amino acids with a short hydrophobic sequence at its N-terminal end which has Ae characteristics of the signal sequences of exported proteins. The signal peptide may be 24 amino acids long, which would corroborate wiA the usual length encountered in prokaryotes. The molecular cloning of the pelZ gene in an expression vector pT7-6 allowed for the specific 35S-cysteine-methionine raAo-labelling of PelZ in E. coli K38. We could detect, in crude extracts, the presence of a precursor and a mature form of PelZ. After cell fractionation, Ae mature form of PelZ could be localized in Ae periplasm of E. coli. So PelZ appears to be a protein exported by Ae Sec-dependent system of translocation. 

PCA Passive cutaneous anaphylaxis pCDM8 Eukaryotic expression vector PCNA Proliferating cell nuclear antigen... 

Figure 7.6. Purification of protein from pooled yeast strains. Each yeast ORF was cloned as a fusion to glutathione-S-transferase in a protein expression vector to create 6144 yeast strains. The individual strains were pooled in groups of 96 to create a set of 64 pools. Each pool was grown and the 96 fusion proteins are purified in batch. Each pool was then assayed for a biochemical function (Martzen et al., 1999). Pools positive for function were then deconvoluted using smaller pools consisting of strains from rows and columns of a 96-well plate.

Besides the standard vectors for constitutive and inducible expression of genes in P. pastoris, new expression vectors and promoters with new regulatory features allowing, for example, also methanol-free inducible high-level expression were discovered or developed by mutagenesis of the mostly used alcohol oxidase promoter Paoxi [87]. Thus, a new P. pastoris expression system became available, which is commercialized by the company VTU (AT). 

Philipps, B., Forstner, M. and Mayr, L.M. (2005) A baculovirus expression vector system for simultaneous protein expression in insect and mammalian cells. Biotechnology Progress, 21 (3), 708-711. 

Hansson, M., Samuelson, P., Nguyen, T.N. and Stahl, S. (2002) General expression vectors for Staphylococcus carnosus enabled efficient production of the outer membrane protein A of Klebsiella pneumoniae. FEMS Microbiology Letters, 210 (2), 263—270. 

Ghosh, S., Parvez, M.K., Banerjee, K. et al. (2002) Baculovirus as mammalian cell expression vector for gene therapy an emerging strategy. Molecular Therapy The Journal of the American Society of Gene Therapy, 6(1), 5-11. 

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