Extracellular secretion of Escherichia coli alkaline phosphatase with a C-terminal tag by type I secretion system: purification and biochemical characterization.
Type I secretion system (TISS) of Gram-negative bacteria permits proteins to be secreted directly from the cytoplasm to the external medium by a single, energy-coupled step. To examine whether this system can be used as an extracellular production system of recombinant proteins, Escherichia coli alkaline phosphatase (AP) was fused to a C-terminal region of Pseudomonas sp. MIS38 lipase (PML) and examined for secretion using the E.coli cells carrying the heterologous TISS. PML is one of the passenger proteins of TISS and contains 12 repetitive sequences and a secretion signal at the C-terminal region. The fusion protein was efficiently secreted to the extracellular medium, while AP was not secreted at all, indicating that the secretion of AP is promoted by a secretion signal of PML. The repetitive sequences were not so important for secretion of the fusion protein, because the secretion level of the fusion protein containing entire repeats ( approximately 10 mg/l culture) was only 2-fold higher than that of the fusion protein without repeats. The fusion protein purified from the culture supernatant existed as a homodimer, like AP, and was indistinguishable from AP in enzymatic properties and stability.
Angkawidjaja
C
C
Department of Material and Life Science, Graduate School of Engineering, Osaka University 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
Kuwahara
K
K
Omori
K
K
Koga
Y
Y
Takano
K
K
Kanaya
S
S
D000469:Alkaline Phosphatase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism*; D008049:Lipase / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D008049:Lipase; D000469:Alkaline Phosphatase