Description

Description: NZYEasy Cloning & Expression kits were designed to allow directional cloning of any PCR-generated fragment or synthetic gene into a linearized pHTP3 Escherichia coli expression vector. Cloning proceeds in a single ligase-independent reaction mediated by the NZYEasy enzyme mix. Vector-complementary overhangs containing a specific sequence recognized by the NZYEasy enzyme are incorporated in the PCR product by using primers with appropriate 5' extensions. When you combine the insert thus generated with the linearized pHTP vector, also containing complementary overhangs, in the presence of NZYEasy enzyme mix, the two DNA molecules will anneal through base-pair complementation of the single-strand regions. The reaction occurs in a single-tube along three temperature-dependent steps. Circular recombinant vector containing the fragment of interest is obtained by transforming the annealed plasmid DNA into competent E. coli cells. The system allows achieving high cloning efficiency (80-100%) and does not require the use of DNA ligases. In addition, the insert does not require any preliminary treatment (e.g. restriction digestion, phosphorylation, or blunt-end polishing). Cloning is performed using conventional E. coli strains. Once pHTP3 recombinant plasmid has been constructed and its sequence confirmed it should be used to transform λDE3 E. coli lysogens, such as BL21(DE3), for high levels of protein expression. NZYEasy Cloning & Expression kits have been successfully used in high-throughput (HTP) platforms for the efficient cloning and expression of a large number of genes at a scale compatible with the functional screen of hundreds to thousands of genes/proteins. NZYTech provides a comprehensive portfolio of pHTP expression vectors, which include different fusion tags commonly used to enhance expression and/or solubility of recombinant proteins in E.coli, as well as fluorescent tags. pHTP1 vector (included in NZYEasy Cloning & Expression kit I, cat. No. MB281) contains two poly-histidine (6xHis) sequences (N- and C-terminal) which allow subsequent recombinant protein purification by immobilized metal ion affinity chromatography (IMAC). The other pHTP expression vectors were constructed by inserting fusion tags (see Table 1) into the pHTP1 backbone such that the fusion partner will be at the N-terminus of the recombinant protein.

Additional Text

Short Description

NZYEasy Cloning & Expression kit III was designed to allow directional cloning of any PCR-generated fragment or synthetic gene into the linearized pHTP3 E. coli expression vector (contains mutDsbC as fusion tag for expression of disulfide bond-containing proteins).