New England Biolabs, E1000S, K. lactis Protein Expression Kit

The Related Categories Protein Expression in Yeast,, Protein Expression Applications Protein Expression in Yeast ,, Protein Expression Specification Shipping Notes Ships on dry ice FAQ Q: What systems does NEB offer for protein expression and purification? A: The NEBExpress® MBP Fusion and Purification System (NEB# E8201) takes advantage of the strong Ptac promoter and the translation initiation signals of maltose binding protein (MBP) to enhance solubility and expression levels of a desired protein in E. coli. The resulting product is an MBP fusion protein, which is then purified and isolated in two easy steps: amylose elution followed by TEV Protease cleavage and Ni resin isolation results in a highly pure tag-free target protein The IMPACT™ System (NEB# E6901) utilizes engineered protein splicing elements (inteins) to purify recombinant proteins by a single chitin affinity column. This system distinguishes itself from other protein fusion systems by its ability to separate a recombinant protein from the affinity tag without the use of a protease. In addition, native recombinant proteins possessing a C-terminal thioester can be isolated for applications such as protein semisynthesis and site specific labeling. The K.lactis Protein Expression Kit (NEB# E1000) 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. The PURExpress® In Vitro Protein Synthesis Kit (NEB# E6800) is an E.coli cell-free transcription/translation system reconstituted from purified components. The E.coli transcription/translation factors (except for ribosomes) are His-tagged. Target protein may be expressed from either linear of plasmid DNA templates containing a T7 promoter. The NEBExpress™ Cell-Free E.coli Protein Synthesis System (NEB #E5360) is an extract-based system derived from E. coli cells that have been engineered for high-level in vitro production of recombinant protein from genes cloned downstream of a T7 promoter in either linear or plasmid DNA templates. Efficient isolation of the recombinant target protein may be accomplished by using NEBExpress Ni-NTA Magnetic Beads (NEB #S1423), spin columns (NEB #S1427) or resin (NEB #S1428). NEBExpress™ Ni-NTA Magnetic Beads (NEB #S1423), NEBExpress™ Ni Spin Columns (NEB #S1427) and NEBExpress™ Ni Resin (NEB #S1428) – a portfolio of immobilized Nickel products for the isolation of poly-histidine tagged (His-tagged) proteins from cell lysates or cell-free protein synthesis reactions. Q: I received my kit and don’t see any competent cells or transformation reagent. Where are they? A: The competent cells and transformation reagent are packaged together (NEB #C1001S) and shipped separately from the kit box at -80°C. Please store the competent cells immediately at -80C° upon arrival. The transformation reagent may be stored at -80°C until ready for use, however, once thawed, it should remain at 4°C and not be re-frozen. The kit box (E1000S) should be stored at -20°C. Q: I’ve expressed my protein, now how do I purify it? A: We recommend customers figure this out before beginning with the kit. Tagging secreted proteins with C- or N-terminal polyhistidine sequences is not recommended. In the majority of reported cases, proteins with a tag of six or ten histidine residues will not bind efficiently to nickel columns and are difficult to detect by western analysis using anti-HIS tag antibodies. Conventional purification resins, such as anion-exchange and hydrophobic resins, may be used. Q: I’ve noticed untransformed K. lactis will grow when streaked on the selection media. Is the selection not working? A: K. lactis contains a weakly active native acetamidase which will allow for minimal growth of untransformed cells on selection media. When a transformation is successful, this background growth is quickly outpaced by that of correctly transformed cells, which form large, 1-2 mm diameter colonies. These colonies have been shown to be nearly 100% positive for cassette integration. Q: Where can I find more detailed FAQs for the K. lactis Protein Expression Kit? A: A complete list of K. lactis Protein Expression FAQs can be found here Q: Can you tell me more about the switch from BSA to Recombinant Albumin (rAlbumin) in NEBuffers? A: NEB is excited to announce that we have switched BSA-containing reaction buffers (NEBuffer 1.1, 2.1, 3.1 and CutSmart® Buffer) to Recombinant Albumin-containing buffers (NEBuffer r1.1, r2.1, r3.1 and rCutSmart™ Buffer). We are also in the process of moving all restriction enzyme formulations to contain rAlbumin. We feel that moving away from animal-containing products is a step in the right direction and are able to offer this enhancement at the same price.