Expression of human placental alkaline phosphatase in Escherichia coli.
The human placental alkaline phosphatase gene was subcloned for expression in Escherichia coli. A shortened form of the alkaline phosphatase, lacking 29 C-terminal amino acids which constitute a membrane anchoring domain, is expressed to about 5% of total protein. Most of the enzyme is present in an insoluble form; however, soluble enzyme can be detected in Western blots and activity tests. The protein is located in the periplasm since its signal peptide is cleaved off. Like the wild-type enzyme, recombinant, shortened placental alkaline phosphatase is inhibited by L-phenylalanine. The amount of active enzyme can be increased by addition of magnesium but not zinc to the medium. Using optimized fermentation conditions, 11 kU of soluble and active alkaline phosphatase are produced per liter. Coexpression of potential folding aids like peptidyl-prolyl cis-trans isomerase or disulfide isomerase does not lead to an increase of soluble enzyme, either when overexpressed separately or from an operon.
Beck
R
R
Boehringer Mannheim Research Center, Penzberg, Germany.
Burtscher
H
H
D000469:Alkaline Phosphatase / Q000096:biosynthesis / Q000235:genetics*; D004926:Escherichia coli; D005285:Fermentation; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005822:Genetic Vectors; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D007535:Isomerases / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011257:Pregnancy Proteins / Q000096:biosynthesis / Q000235:genetics*; D019704:Protein Disulfide-Isomerases; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics*
D011257:Pregnancy Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D005947:Glucose