Construction of a Chimeric Thermostable Pyrophosphatase To Facilitate Its Purification and Immobilization by Using the Choline-Binding Tag

Abstract

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.

Mechanism of cell surface expression of the Streptococcus mitis platelet binding proteins PblA and PblB

“…Both PblA and PblB contain repeat regions of glycine‐tryptophan‐rich motifs, similar to the choline binding proteins of S. pneumoniae , and the lipoteichoic acid binding regions of InlB of Listeria monocytogenes (Jonquieres et al ., 1999). Moreover, we have shown that PblA and PblB can be purified from culture media by affinity chromatography with DEAE cellulose (a choline analogue), as has been done with other choline binding proteins (Sanchez‐Beato et al ., 1995; Caubin et al ., 2001; Fan et al ., 2004; Moldes et al ., 2004).…”

Strategies for the one-step immobilization–purification of enzymes as industrial biocatalysts

“…Perhaps the most popular domain is the poly-His tag, with high affinity for metal chelates (Figure 5) (e.g. for purification by IMAC) (Porath et al, 1975, Porath and Olin, 1983, Porath, 1992 but the range of these affinity peptides is huge and still growing: domains of affinity for cellulose, chitin binding domain, peptide tags, among 9 others (Arroyo et al, 2011, Bello-Gil et al, 2014, Bergeron et al, 2009, Bolivar and Nidetzky, 2012a, b, Cassimjee et al, 2011, Chern and Chao, 2005, Daunert et al, 2007, Kondo and Teshima, 1995, Kowsari et al, 2014, Kweon et al, 2005, Linder and Teeri, 1997, Martinez et al, 2000, Mateo et al, 2001b, Moldes et al, 2004a, Moldes et al, 2004b, Scaramozzino et al, 2005, Shpigel et al, 1999, Vishwanath et al, 1995, Wang et al, 2013a, Wiesbauer et al, 2011, Zhao et al, 2013. Table 1 shows a summary of the main domains used for this purpose while Table 2 shows some specific examples of uses of these domains.…”

Streptococcus pneumoniae: from molecular biology to host-pathogen interactions

“…In this sense, analysis of a collection of 21 mutated LytA NAM-amidases indicated that Ile-315, located in the last CBR, is a key amino acid residue in both enzymatic activity and folding (Romero et al 2007). Moreover, it is worth to mention that selective interaction of the CBM of LytA with choline or its analogs (tertiary or quaternary amines) has allowed its use as a tag to construct a variety of fusion proteins that may be purified using DEAE-cellulose, which acts as an affinity matrix for CBM-containing proteins (Sánchez-Puelles et al 1992, Moldes et al 2004. In this sense, a commercial kit based in this system is already available (C-LYTAG; http://www.biomedal.com).…”