pMAL™ System

The pMAL™ Protein Fusion and Purification System is available for producing a protein expressed from a cloned gene or open reading frame. The gene of interest is fused to the malE gene, which encodes maltose-binding protein (MBP), and expressed at levels constituting up to 40% of the total cell protein using the strong tac promoter and translation initiation signals of MBP. A one-step affinity purification for MBP is used to isolate the fusion protein.

IMPACT™ Systems

The IMPACT-CN and IMPACT-TWIN Systems distinguish themselves from other fusion systems by their ability to purify native, recombinant proteins free of an affinity tag after a single chromatographic step. The IMPACT Systems can also be used for protein ligation which allows researchers to assemble toxic proteins in vitro and label a portion of the protein for NMR studies. In addition, cyclic peptides that may be resistant to exopeptidases can also be produced.

The IMPACT Systems were developed based on mechanistic studies of protein splicing. Protein splicing involves the precise excision of an internal protein segment, the intein, from a precursor protein followed by the concomitant ligation of the flanking N- and C-terminal regions, the exteins, yielding two protein products. Investigation into the chemical mechanisms by which inteins break and make peptide bonds has not only helped us understand the extraordinary phenomenon of protein splicing but has also evolved into this novel method of purifying native recombinant proteins in a single affinity purification step.

The gene of interest is cloned into an expression vector to create a target protein-intein-chitin binding domain (CBD) fusion. Expression of the fusion construct is controlled by an IPTG-inducible T7 promoter. The three part fusion protein is bound to chitin beads and the protein of interest is released from the intein-CBD tag after induced on-column cleavage.

K.lactis System

The K.lactis Protein Expression Kit (NEB# E1000S) provides an easy method for expressing a gene of interest in the yeast Kluyveromyces lactis. Proteins may be produced intracellularly or be secreted using the supplied integrative expression vector pKLAC1. To achieve protein secretion, a gene of interest is cloned downstream of the K. lactis a-mating factor secretion domain which is eventually processed in the Golgi resulting in the secretion of the desired protein. K. lactis rapidly achieves high culture densities and abundantly produces recombinant proteins. Yeast expression is driven by a variant of the strong LAC4 promoter that has been modified to lack background expression in E.coli. Therefore, genes toxic to E.coli can be cloned into pKLAC1 in bacteria prior to their expression in yeast. The high transfomation efficiency of the supplied K.lactis cells makes the system suitable for methods that require large numbers of transformants, for example, expression cloning using cDNA libraries. Selection of yeast transformants uses a unique antibiotic-free method in which acetamidase expressed form pKLAC1 permits transformed cells to utilize acetamide as a sole nitrogen source on defined medium. Acetamide selection promotes formation of cells containing multiple integrations of pKLAC1 which results in higher yields of protein.

RheoSwitch® System

The RheoSwitch® Mammalian Inducible Expression System represents the next generation of inducible gene expression systems for mammalian cells. Analogous to the operation of a rheostat, the RheoSwitch technology allows induction and adjustable control of gene expression. Regulation of gene expression is achieved through the highly specific interaction of a synthetic inducer, RheoSwitch Ligand RSL1, and a chimeric bipartite nuclear receptor. This receptor is activated in the presence of RSL1 ligand, and the level of gene expression can be regulated by adjusting the concentration of RSL1 ligand in the tissue culture media. The precise control of the RheoSwitch technology is unrivaled among mammalian expression systems, giving negligible levels of basal expression in the absence of inducer and greater than 10,000 fold induction when RSL1 ligand is present.