69954  pET-31b(+) DNA, AlwN I Digest

This kit is available as a convenient supplement to the pET Peptide Expression System 31b, which is designed for expression and purification of target peptides (1). In pET-31b(+), peptide coding sequences are placed downstream of a 125 amino acid (aa) ketosteroid isomerase (KSI) gene and upstream of a His•Tag^® sequence. Use of the unique AlwN I cloning site enables unidirectional insertion of coding sequences immediately adjacent to a methionine residue. For high-yield production, small (10–25 aa) peptide coding sequences can be cloned as tandem repeats interspersed with single methionine residues. The KSI-peptide-His•Tag fusion protein is expressed at high levels in the presence of T7 RNA polymerase provided by the recA^–, protease deficient BLR(DE3)pLysS host. The fusion protein is purified in one step under denaturing conditions using one of Novagen's His•Bind^® metal chelation resins. It is then cleaved with CNBr to release the His•Tag peptide, insoluble KSI, and monomer peptide units each terminating with a homoserine lactone. The purified peptide can be easily converted to an amide or homoserine, or reacted with other compounds, such as analogs of fluorescein and biotin, to produce C-terminal derivatized peptides. The system has been used to make biologically active peptides of 14–67 aa at yields of > 50 mg purified peptide per liter of culture (1).

The AlwN I digested, dephosphorylated vector is ready to accept appropriate inserts containing ...ATG-3′ and 3′-TAC... overhangs on the top and bottom strands, respectively. A Control Insert is provided to verify performance and ensure optimal cloning efficiency.

Features
• High yield expression of peptides and small proteins
• His•Tag sequence facilitates protein purification
• Prepared vector facilitates cloning with minimal background
• Special host strain inhibits degradation of expressed proteins..

This product is covered under license from Brookhaven National Labs. Commercial entities need to obtain a research use license prior to purchase. Please contact your licensing department to confirm that your company already holds a research use license.