pADL-pIII-2 Vector

The pADL-pIII-2 vector is a specially designed phagemid that's used for phage display on the N-terminal side of protein III in bacteriophage M13 or equivalent phage. To start, the pADL-pIII-2 vector contains a PelB leader sequence. This allows for enhanced expression in the periplasm, a space located between the outer and inner membranes in gram-negative bacteria, where many important biochemical processes occur. For inserting single-chain variable fragments (scFvs) or Fab fragments, the vector is armed with a double-SfiI cloning site. These fragments represent the active sites of antibodies and can be used for a wide array of applications, including detection, targeting, and therapeutic strategies. Furthermore, the pADL-pIII-2 vector includes a His tag or hexahistidine tag, which enables purification of the protein of interest that is overexpressed in the bacteria. This tag allows for simple and efficient affinity purification under native or denaturing conditions using immobilized metal ion affinity chromatography (IMAC). Additionally, the vector encompasses an HA tag for detection, which is a short peptide sequence from the influenza hemagglutinin protein that is widely used as an epitope tag. An Amber codon is located before the complete copy of the gene III sequence. This codon stops translation in non-suppressor strains, producing free scFvs or Fab fragments in the periplasm, where they can be assessed for binding or purified for subsequent testing. The expression of the fusion genetic material within the pADL-pIII-2 vector is firmly regulated by a LacZ promoter. This promoter is negatively regulated, meaning it is switched off in the presence of glucose and is only active when lactose or an analog like IPTG is present. A potent transcriptional terminator situated upstream of the promoter efficiently hampers undesirable expression and prevents promoter leakiness in the absence of induction, which in turn restricts negative selection against clones encoding toxic products.