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.
Moldes
Cristina
C
Department of Molecular Microbiology, Centro de Investigaciones Biológicas, Ramiro de Maeztu 9, 28040 Madrid, Spain.
García
José L
JL
García
Pedro
P
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