Immobilization and single‐step purification of fusion proteins using DEAE‐cellulose

Abstract

We have developed a new single-step system, using a DEAE matrix, to immobilize and/or purify fusion proteins containing the choline-binding domain of the Streptococcus pneumoniae murein hydrolases. We have constructed a choline-binding-domain -P-galactosidase chimera, which can be purified by this procedure and shows a high P-galactosidase activity when immobilized in the column. A vector plasmid, pCUZl, containing the Ippp-5/lac promoter as well as 13 restriction sites, was constructed to facilitate the cloning and expression of gene fusions. This plasmid also allows the selection of recombinants by the well-known blue/white 5-bromo-4-chloro-3-indolyl-~-~-galactoside procedure.

Structural basis of PcsB-mediated cell separation in Streptococcus pneumoniae

“…The extended CC PcsB is composed of residues arranged in a continuous helical arrangement. Only the first four residues (41)(42)(43)(44)(45) and those of the short turns do not follow this pattern. All the helices within the CC domain are located in the same plane except for the a5 helix.…”

“…To construct the pcsB-CHAP lytF chimeric gene, pneumococcal pcsB without its CHAP-encoding part (bp 1-858) was amplified using the primer pair 216/52 and genomic DNA from strain RH1 (ref. 41) as template. The CHAP-encoding part of lytF (bp 1260-1578) was amplified using the primer pair 53/217 and genomic DNA from S. gordonii (Challis) as template.…”

Functional Analysis of the Small Component of the 4-Hydroxyphenylacetate 3-Monooxygenase of Escherichia coli W: a Prototype of a New Flavin:NAD(P)H Reductase Subfamily

“…In fact, we have detected a low FAD-dependent reductase activity (5 nmol min Ϫ1 mg Ϫ1 ) in HpaB preparations. Therefore, to purify the HpaB enzyme by a faster and more selective procedure, we constructed a chimeric tagged HpaB protein (CHHpaB) by fusing the choline-binding domain of the major autolysin of Streptococcus pneumoniae (35) to the N terminus of HpaB (Fig. 2).…”

“…2). The CH-HpaB protein can be purified free of reductase-contaminating activities in a single step by affinity chromatography with DEAE-cellulose, a choline analoguecontaining matrix (35). E. coli TG1 cells harboring plasmid pAJ31 produced large amounts of the CH-HpaB fusion both as soluble and insoluble (inclusion bodies) protein.…”

The lytic enzyme of the pneumococcal phage Dp‐1: a chimeric lysin of intergeneric origin

“…The Pal enzyme was purified from extracts of DH5␣ (pMSP11) by affinity chromatography in DEAE-cellulose, as described previously for choline-binding proteins (Sá nchez-Puelles et al, 1992). Briefly, cells were grown in LB medium supplemented with ampicillin (100 g ml ¹1 ) and 0.4 mM IPTG at 37ЊC for 24 h with shaking, harvested by centrifugation and broken in a French press cell.…”