Expression of recombinant proteins with uniform N-termini.
Heterologously expressed proteins in Escherichia coli may undergo unwanted N-terminal processing by methionine and proline aminopeptidases. To overcome this problem, we present a system where the gene of interest is cloned as a fusion to a self-splicing mini-intein. This fusion construct is expressed in an engineered E. coli strain from which the pepP gene coding for aminopeptidase P has been deleted. We describe a protocol using human cationic trypsinogen as an example to demonstrate that recombinant proteins produced in this expression system contain homogeneous, unprocessed N-termini.
Department of Molecular and Cell Biology, Henry M. Goldman School of Dental Medicine, Boston University, Boston, MA, USA. okiraly@mit.edu
Orsolya
O
Király
Lan
L
Guan
Miklós
M
Sahin-Tóth
D000626:Aminopeptidases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics; D017353:Gene Deletion; D006801:Humans; D047668:Inteins; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D014362:Trypsinogen / Q000096:biosynthesis* / Q000235:genetics
D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D014362:Trypsinogen; D000626:Aminopeptidases; C531476:aminopeptidase P, E coli