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Expression of human cationic trypsinogen with an authentic N terminus using intein-mediated splicing in aminopeptidase P deficient Escherichia coli.


ABSTRACT: 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.

SUBMITTER: Kiraly O 

PROVIDER: S-EPMC1604731 | biostudies-literature | 2006 Jul

REPOSITORIES: biostudies-literature

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Expression of human cationic trypsinogen with an authentic N terminus using intein-mediated splicing in aminopeptidase P deficient Escherichia coli.

Király Orsolya O   Guan Lan L   Szepessy Edit E   Tóth Miklós M   Kukor Zoltán Z   Sahin-Tóth Miklós M  

Protein expression and purification 20060221 1


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 splicin  ...[more]

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