Detection and isolation of recombinant protein based on binding affinity reporter. Maltose binding protein.

Abstract

AbstractThe pMAL-2 vectors (Fig. 1) provide a method for expressing and purifying a protein produced from a cloned gene or open reading frame. The cloned gene is inserted downstream from the malE gene of E. coli, which encodes maltose-binding protein (MBP), resulting in the expression of an MBP fusion protein (1,2). The method uses the strong “tat” promoter and the malE translation initiation signals to give high-level expression of the cloned sequences (3,4), and a one-step purification of the fusion protein using MBP’s affinity for maltose (5). The vectors express the malE gene (with or without its signal sequence) fused to the lacZa gene. Restriction sites between malE and lacZα are available for inserting the coding sequence of interest. Insertion inactivates the β-galactosidase α-fragment activity of the malE-lacZa fusion, which results in a blue to white color change on X-gal plates when the construction is transformed into an α-complementing host such as TB1 (6) or JM107 (7). The vectors carry the lacI q gene, which codes for the Lac repressor. This keeps expression from Ptac low in the absence of IPTG (isopropyl-β-D-thiogalactoside) induction. The pMAL-2 vectors also contain the sequence coding for the recognition site of the specific protease factor Xa (9,10), located Just 5′ to the polylinker insertion sites. This allows MBP to be cleaved from the protein of interest after purification. Factor Xa cleaves after its four amino acid recognition sequence, so that few or no vector-derived residues are attached to the protein of interest, depending on the site used for cloning. A purification example is shown in Fig. 2. pMAL-2 vectors are shown pMAL-c2 (6646 bp) has an exact deletion of the malE signal sequence pMAL-p2 (6721 bp) includes the malE signal sequence. Arrows indicate the direction of transcription. Unique restriction sites are indicated. Flowchart for the pilot experiment. KeywordsFusion ProteinPeriplasmic FractionFusion PlasmidColumn BufferAmylose ResinThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.