Construction of a chimeric thermostable pyrophosphatase to facilitate its purification and immobilization by using the choline-binding tag.
The thermophilic inorganic pyrophosphatase (Pyr) from Thermus thermophilus has been produced in Escherichia coli fused to the C terminus of the choline-binding tag (ChB tag) derived from the choline-binding domain (ChBD) of pneumococcal LytA autolysin. The chimeric ChBD-Pyr protein retains its thermostable activity and can be purified in a single step by DEAE-cellulose affinity chromatography. Pyr can be further released from the ChBD by thrombin, using the specific protease recognition site incorporated in the C terminus of this tag. Remarkably, the ChB tag provides a selective and very strong thermostable noncovalent immobilization of ChBD-Pyr in the DEAE-cellulose matrix. The binding of choline or choline analogues, such as DEAE, confers a high thermal stability to this tag; therefore, the immobilized chimeric enzyme can be assayed at high temperature without protein leakage, demonstrating the usefulness of the ChB tag for noncovalent immobilization of thermophilic proteins. Moreover, ChBD-Pyr can be purified and immobilized in a single step on commercial DEAE-cellulose paper. The affinity of the ChB tag for this versatile solid support can be very helpful in developing many biotechnological applications.
Department of Molecular Microbiology, Centro de Investigaciones Biológicas, Ramiro de Maeztu 9, 28040 Madrid, Spain.
Cristina
C
Moldes
José L
JL
García
Pedro
P
García
D001483:Base Sequence; D001709:Biotechnology / Q000379:methods; D002794:Choline / Q000378:metabolism*; D004795:Enzyme Stability; D004800:Enzymes, Immobilized; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006358:Hot Temperature; D008969:Molecular Sequence Data; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000737:chemistry; D011755:Pyrophosphatases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013824:Thermus / Q000201:enzymology*
D004800:Enzymes, Immobilized; D011993:Recombinant Fusion Proteins; D009238:N-Acetylmuramoyl-L-alanine Amidase; D011755:Pyrophosphatases; D002794:Choline