Fusions of secreted proteins to alkaline phosphatase: an approach for studying protein secretion.
We have constructed a series of plasmids containing a modified form of the phoA gene of Escherichia coli K-12 that have general utility for studies of protein secretion. In these plasmids, the promoter and signal sequence-encoding region of the phoA gene have been deleted; thus, expression of the gene, giving rise to active alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1], is absolutely dependent upon fusion in the correct reading frame to DNA containing a promoter, a translational start site, and a complete signal sequence-encoding region. Alkaline phosphatase, which is normally located in the periplasm of E. coli, is efficiently secreted to the periplasm when fused either to a signal sequence from another periplasmic protein, beta-lactamase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6), or to signal sequences from the outer membrane proteins LamB and OmpF. These heterologous signal sequences are processed during secretion. In the absence of a complete signal sequence, phosphatase becomes localized in the cytoplasm and is inactive. Phosphatase fusion proteins lacking up to 13 amino-terminal amino acids beyond the signal sequence show the same specific activity as that of the wild-type enzyme. However, a significant decrease in activity is seen when 39 or more amino-terminal amino acids are deleted. Addition of approximately 150 amino acids from the enzyme beta-lactamase to the amino terminus of alkaline phosphatase has little effect on the specific activity of the enzyme. The ability to change the amino terminus of phosphatase without altering its activity makes the enzyme particularly useful for construction of protein fusions. The fact that phosphatase is designed for transport across the cytoplasmic membrane makes it an ideal tool for study of protein secretion.
D000469:Alkaline Phosphatase / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D001692:Biological Transport; D005818:Genetic Engineering; D008566:Membranes / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D010957:Plasmids; D011498:Protein Precursors / Q000378:metabolism*; D021382:Protein Sorting Signals; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D010455:Peptides; D011498:Protein Precursors; D021382:Protein Sorting Signals; D000469:Alkaline Phosphatase