Independently expressed N-terminal pro-domain of aqualysin I precursor complements the folding of its mature domain to active form in Escherichia coli.
Aqualysin I is a subtilisin-type serine protease secreted into the culture medium by Thermus aquaticus YT-1. It is first produced as a large precursor that consists of a signal peptide, an N-terminal pro-domain, the mature protease domain and a C-terminal pro-domain. To investigate whether the N-terminal pro-domain supplied in trans as an independent peptide plays an important role in the folding and secretion of the protease, the N-terminal pro-domain in E. coli has been expressed independent of the mature domain with or without the C-terminal pro-domain using an expression system with separate promoters and signal peptides. Protease assay and SDS-PAGE clearly showed that the N-terminal pro-domain plays an essential role in guiding the proper folding in trans of the enzymatically active conformation of aqualysin I. The N-terminal amino acid sequences of the purified enzymes were identical and had no signal peptides. These results indicate that independently expressed domains are secreted into the periplasmic space before the N-terminal pro-domain-assisted folding of the mature domain.
Divison of Biotechnology, Faculty of Natural Resources and Life Science, Dong-A University, Busan 604-714, Korea.
Ji-Youn
JY
Kim
Yong-Lark
YL
Choi
Young-Su
YS
Cho
Cherol-Ho
CH
Kim
Young-Choon
YC
Lee
D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011401:Promoter Regions, Genetic; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry* / Q000378:metabolism*; D021382:Protein Sorting Signals; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
D011498:Protein Precursors; D021382:Protein Sorting Signals; D012697:Serine Endopeptidases; C055102:aqualysin I