Purification and renaturation of Dictyostelium recombinant alkaline phosphatase by continuous elution electrophoresis.
A 1583 bp fragment of Dictyostelium alp cDNA (94% of the gene) was cloned in pET32a+. The enzyme was expressed in an inactive form in the inclusion body of the expression host BL21-CodonPlus (DE3)-RIL. The recombinant ALP constituted more than 50% of the total protein in the inclusion body and 25-30% of the total protein in the expression host after 3 h induction with IPTG at 37 degrees C. A continuous elution polyacrylamide gel electrophoresis procedure was used to purify the recombinant enzyme. This technique yielded a homogeneous protein that retained enzymatic activity after dialysis without further treatment. A yield of 5mg per liter of culture broth was obtained with a specific activity of approximately 0.7 nmol/min/mg protein (0.7 mU/mg). Immunoinhibition studies using a polyclonal antibody produced against the recombinant protein showed complete inhibition of enzymatic activity when the enzyme was preincubated with the antibody at a 1:1000 dilution. The enzyme exhibited a pH optimum of approximately 9.0. The substrate specificity indicated that the Dictyostelium enzyme is a typical broad range alkaline phosphatase.
Ubeidat
Muatasem
M
Department of Biology, Molecular and Cellular Biology Section, Virginia Polytechnic Institute and State University, 2119 Derring Hall, Blacksburg, VA 24061-0406, USA.
Rutherford
Charles L
CL
D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D003062:Codon; D018076:DNA, Complementary / Q000378:metabolism; D004023:Dictyostelium / Q000201:enzymology*; D004586:Electrophoresis / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D008957:Models, Genetic; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013997:Time Factors
D003062:Codon; D018076:DNA, Complementary; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase