Expression of human cationic trypsinogen with an authentic N terminus using intein-mediated splicing in aminopeptidase P deficient Escherichia coli.
High-level expression of human trypsinogens as inclusion bodies in Escherichia coli requires deletion of the secretory signal sequence and placement of an initiator methionine at the N terminus. Trypsinogen preparations obtained this way contain a mixture of abnormal N termini, as a result of processing by cytoplasmic aminopeptidases. Here, we describe an expression system that produces recombinant human cationic trypsinogen with a native, intact N terminus, using intein-mediated protein splicing and an aminopeptidase P (pepP) deficient E. coli strain. As a first application of this system, the effect of the pancreatitis-associated mutation A16V on the autoactivation of human cationic trypsinogen was characterized. The use of the novel pepP knock-out E. coli strain should be generally applicable to the expression of recombinant proteins, which undergo unwanted N-terminal trimming by aminopeptidase P.
Department of Molecular and Cell Biology, Goldman School of Dental Medicine, Boston University, Boston, MA 02118, USA.
Orsolya
O
Király
Lan
L
Guan
Edit
E
Szepessy
Miklós
M
Tóth
Zoltán
Z
Kukor
Miklós
M
Sahin-Tóth
D000626:Aminopeptidases / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D047668:Inteins; D008969:Molecular Sequence Data; D009154:Mutation; D019154:Protein Splicing; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D014357:Trypsin; D014362:Trypsinogen / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014362:Trypsinogen; D000626:Aminopeptidases; C022750:X-Pro aminopeptidase; C489303:PRSS1 protein, human; D014357:Trypsin