title: Construction of an expression system for aqualysin I in Escherichia coli that gives a markedly improved yield of the enzyme protein.
abstract: An expression system for aqualysin I from Thermus aquaticus YT-1, a thermophilic serine protease belonging to the proteinase K family, in Escherichia coli is available, but the efficiency of production has been rather low for detailed analysis of the product. We developed a maltose biding protein (MBP)-fused proaqualysin I expression plasmid (pMAQ-c2Delta) in which MBP is attached to the N-terminus of proaqualysin I. MBP appeared effectively to suppress the folding-promoting activity of the N-terminal propeptide when the bacteria were grown at 30 degrees C, leading to a massive accumulation of fusion aqualysin I precursor. The precursor was converted efficiently to mature aqualysin I by heat treatment at 70 degrees C, enabling us to obtain 40 times more aqualysin I than is available using expression systems such as pAQNDeltaC105. By analyzing the product of the pMAQ-c2Delta-derived inactive mutant expression vector, pMAQ-S222A, it was confirmed that aqualysin I was initially expressed as a whole fusion protein and then processed autocatalytically.
authors.affiliation: Department of Applied Chemistry, Kogakuin University, Hachioji, Tokyo, Japan.
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Sakaguchi
authors.forename: Keisuke
authors.initials: K
authors.lastname: Niimiya
authors.forename: Makoto
authors.initials: M
authors.lastname: Takezawa
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Toki
authors.forename: Yasusato
authors.initials: Y
authors.lastname: Sugahara
authors.forename: Masao
authors.initials: M
authors.lastname: Kawakita
mesh_terms: D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; C055102:aqualysin I