pDAP2: a vector for construction of alkaline phosphatase fusion-proteins.
BACKGROUND
Expression of enzymatically active protein fusions in Escherichia coli could facilitate the analysis of proteins and even replace some reagents frequently used in immunology such as chemically produced antibody-enzyme conjugates. For this purpose there is up to now no system of general utility available.


OBJECTIVE
The vector pDAP2 has been designed for simplified fusion of single-chain Fv fragments to the N-terminus of E. coli alkaline phosphatase. The resulting immunoconjugates can be produced by expression in E. coli and purified in a single step via metal affinity chromatography.


METHODS
Several different single-chain Fv genes as well as peptide coding oligonucleotides have been cloned into pDAP2 and tested for expression levels, purification properties and usefulness in ELISA and immunowestern blotting.


RESULTS
The fusion proteins from pDAP2 can be prepared at levels of several milligrams per liter culture from the periplasma of the cells. The proteins work well in different immunoassay formats such as ELISA or western blotting.


CONCLUSIONS
pDAP2 is compatible to phage display vectors such as pHEN1, pCOCK and the pCANTAB-series (Pharmacia, Sweden). Genes of single-chain antibody fragments can be simply swapped from these vectors into pDAP2. Furthermore, we demonstrate that it is possible to produce alkaline phosphatase-active peptides with this vector by inserting synthetic coding oligonucleotides. This allows simple analysis of the binding properties of peptides and may have some advantages over other systems such as fusion to glutathione-S-transferase.
Kerschbaumer
R J
RJ
Institute of Applied Microbiology, University of Agriculture and Forestry, Vienna, Austria.
Hirschl
S
S
Schwager
C
C
Ibl
M
M
Himmler
G
G
D000469:Alkaline Phosphatase / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins
D007128:Immunoglobulin Fragments; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv; D000469:Alkaline Phosphatase