pNMT-TOPO 酵母表达载体 BioVector NTCC质粒载体菌种细胞基因保藏中心

pNMT-TOPO 酵母表达载体

BioVector NTCC质粒载体菌种细胞基因保藏中心                          

The pNMT TOPO® TA Expression Kits use TOPO® Cloning technology to provide a highly efficient, rapid cloning strategy for the direct insertion of Taq polymeraseamplified PCR products into a series of plasmid vectors for regulated expression of the gene of interest in the fission yeast, Schizosaccharomyces pombe (S. pombe). TOPO® Cloning requires no ligase, post-PCR procedures, or PCR primers containing special, additional sequences. Once cloned, analyzed, and transformed into an S. pombe host strain, expression of the PCR product can be regulated using thiamine.The pNMT1-TOPO®, pNMT41-TOPO®, and pNMT81-TOPO® vectors are 6.1 kb expression vectors designed to facilitate rapid cloning and thiamine-regulated expression of PCR products using the SpECTRA™ S. pombe Expression System (Catalog no. K180- 01) available from Invitrogen. The pNMT-TOPO® vectors contain the following elements: • The nmt1 promoter for thiamine-regulated expression of the gene of interest in S. pombe cells (Maundrell, 1990). Each vector contains a distinct form of the nmt1 promoter differing only in the TATA box region (see page 3 for details) (Basi et al., 1993). • TOPO® Cloning site for rapid and efficient cloning of Taq-amplified PCR products (see the next page for more information) • C-terminal peptide containing the V5 epitope and a polyhistidine (6xHis) tag for detection and purification of recombinant protein (optional) • S. pombe ars1 origin of replication for non-integrative, high-copy maintenance of the plasmid in S. pombe cells (Heyer et al., 1986) • S. cerevisiae LEU2 auxotrophic marker for selection of yeast transformants (Andreadis et al., 1984) • Ampicillin resistance gene for selection in E. coli.The plasmid vectors, pNMT1-TOPO®, pNMT41-TOPO®, and pNMT81-TOPO®, are supplied linearized with: • Single 3´ thymidine (T) overhangs for TA Cloning® • Topoisomerase covalently bound to the vector (this is referred to as “activated” vector) Taq polymerase has a nontemplate-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3´ ends of PCR products. The linearized vector supplied in this kit has single, overhanging 3´ deoxythymidine (T) residues. This allows PCR inserts to ligate efficiently with the vector. Topoisomerase I from Vaccinia virus binds to duplex DNA at specific sites and cleaves the phosphodiester backbone after 5′-CCCTT in one strand (Shuman, 1991). The energy from the broken phosphodiester backbone is conserved by formation of a covalent bond between the 3′ phosphate of the cleaved strand and a tyrosyl residue (Tyr-274) of topoisomerase I. The phospho-tyrosyl bond between the DNA and enzyme can subsequently be attacked by the 5′ hydroxyl of the original cleaved strand, reversing the reaction and releasing topoisomerase (Shuman, 1994). TOPO® Cloning exploits this reaction to efficiently clone PCR productsnmt1 Promoter and Thiamine Regulation In S. pombe, the no message in thiamine (nmt1) gene encodes a 39 kDa protein that is both regulated by thiamine and thought to be involved in its biosynthesis (Maundrell, 1990). The nmt1 gene and its promoter have been well-studied and found to exhibit the following characteristics: • In minimal medium, the nmt1 gene is highly transcribed (Maundrell, 1990). • In minimal medium supplemented with thiamine, expression of the nmt1 gene is repressed (Maundrell, 1990). • Complete repression of transcription occurs when thiamine is added to a final concentration of 0.5 µM or greater (Maundrell, 1990). • Addition of thiamine to the culture medium results in the disappearance of message within 3 hours (Maundrell, 1990): • Removal of thiamine results in approximately 100-200-fold induction of expression from the nmt1 promoter. • Removal of thiamine results in detectable message after 10 hours and maximal, steadystate levels after 16 hours (Maundrell, 1990). The SpECTRA™ S. pombe Expression System uses the nmt1 promoter to impart thiamineregulation to a heterologous gene of interest. In the system, expression of your gene of interest is repressed in the presence of thiamine and induced in the absence of thiamine. In general, the kinetics of heterologous gene expression are similar to those described above with the native nmt1 gene.TATA Box Mutations in the nmt1 Promoter Detailed analysis of the nmt1 promoter has demonstrated that it contains a canonical TATA box element located 25 base pairs upstream of the transcriptional start site (Maundrell, 1990) (see the diagram on page 6). Further studies have shown that stepwise truncation of the TATA box leads to a progressive decrease in the strength of the nmt1 promoter (Basi et al., 1993). Both the induced level and the basal, repressed level of the nmt1 gene (and heterologous genes) are reduced (Basi et al., 1993). The pNMT1-TOPO®, pNMT41-TOPO®, and pNMT81-TOPO® vectors contain the wildtype nmt1 promoter and two TATA box mutants, respectively (see figure below). TOPO® Cloning your gene of interest into pNMT1-TOPO®, pNMT41-TOPO®, and pNMT81- TOPO® allows the user to vary the range of thiamine-regulated expression of the recombinant protein of interest.      [Supplier来源]  http://www.biovector.net