title: Cloning strategy, production and purification of proteins with exopeptidase-cleavable His-tags.
abstract: Here, we present a cloning strategy for the production of recombinant proteins tagged with a polyhistidine sequence that can be cleaved by the exopeptidase, DAPase. The method can be used with most commonly available vectors and results in the expression of a His-tag protein that can be purified in its native form regardless of its natural sequence. This approach takes advantage of the TAGZyme system for the removal of amino-terminal affinity tags. Tag removal is accomplished either with DAPase (a recombinant dipeptidyl peptidase) alone or in combination with two accessory enzymes, Qcyclase and pGAPase. The system has been used for the production of intracellular proteins in Escherichia coli and can be applied to other expression hosts for the production of secreted proteins or proteins that require post-translational modification. The production of human interleukin 1beta in E. coli is used as an example to illustrate this method. The complete protocol from initial PCR to the production of a detagged protein with its authentic N terminus can be performed within 5 days.
authors.affiliation: Unizyme Laboratories, Dr Neergaards vej 17, DK 2970 Hørsholm, Denmark. ja@unizyme.dk
authors.forename: José
authors.initials: J
authors.lastname: Arnau
authors.forename: Conni
authors.initials: C
authors.lastname: Lauritzen
authors.forename: John
authors.initials: J
authors.lastname: Pedersen
mesh_terms: D000345:Affinity Labels / Q000378:metabolism; D020816:Amino Acid Motifs; D020789:Cathepsin C / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism*; D053583:Interleukin-1beta / Q000096:biosynthesis* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
chemical_list: D000345:Affinity Labels; D053583:Interleukin-1beta; D011994:Recombinant Proteins; C033223:polyhistidine; D006639:Histidine; D020789:Cathepsin C