In vitro stepwise autoprocessing of the proform of pro-aminopeptidase processing protease from Aeromonas caviae T-64

Bing Tang, Satoru Nirasawa, Motomitsu Kitaoka, Kiyoshi Hayashi
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引用次数: 13

Abstract

PA protease (pro-aminopeptidase processing protease) is an extracellular zinc metalloprotease produced by the Gram-negative bacterium Aeromonas caviae T-64. The 590-amino-acid precursor of PA protease is composed of a putative 19-amino-acid signal sequence, a 165-amino-acid N-terminal propeptide, a 33 kDa mature protease domain and an 11 kDa C-terminal propeptide. The proform of PA protease, which was produced as inclusion bodies in Escherichia coli, was subjected to in vitro refolding. It was revealed that the processing of the proform involved a stepwise autoprocessing mechanism. Firstly, the N-terminal propeptide was autocatalytically removed on completion of refolding and secondly, the C-terminal propeptide was autoprocessed after the degradation of the N-terminal propeptide. Both the N- and C-terminal propeptides existed as intact peptides after their successive removal, and they were subsequently degraded gradually. The degradation of the N-terminal propeptide appears to be the rate-limiting step in the maturation of the proform of PA protease.

洞穴气单胞菌T-64前氨基肽酶加工蛋白酶形态的体外分步自动加工
PA蛋白酶(原氨基肽酶加工蛋白酶)是由革兰氏阴性细菌气单胞菌T-64产生的细胞外锌金属蛋白酶。PA蛋白酶的590个氨基酸前体由一个19个氨基酸的信号序列、一个165个氨基酸的n端前肽、一个33 kDa的成熟蛋白酶结构域和一个11 kDa的c端前肽组成。在大肠杆菌中以包涵体形式产生的PA蛋白酶形式进行了体外再折叠。结果表明,成形过程中涉及到一种逐步自动加工机制。首先,n端前肽在完成重折叠时被自动催化去除,其次,n端前肽降解后,c端前肽被自动加工。N端前肽和c端前肽在连续去除后作为完整肽存在,随后逐渐降解。n端前肽的降解似乎是PA蛋白酶形式成熟的限速步骤。
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