Phage Display of an Intracellular Carboxylesterase of Bacillus subtilis : Comparison of Sec and Tat Pathway Export Capabilities

Abstract

Using the phage display technology, a protein can be displayed at the surface of bacteriophages as a fusion to one of the phage coat proteins. Here we describe development of this method for fusion of an intracellular carboxylesterase of Bacillus subtilis to the phage minor coat protein g3p. The carboxylesterase gene was cloned in the g3p-based phagemid pCANTAB 5E upstream of the sequence encoding phage g3p and downstream of a signal peptide-encoding sequence. The phage-bound carboxylesterase was correctly folded and fully enzymatically active, as determined from hydrolysis of the naproxen methyl ester with K m values of 0.15 mM and 0.22 mM for the soluble and phage-displayed carboxylesterases, respectively. The signal peptide directs the encoded fusion protein to the cell membrane of Escherichia coli, where phage particles are assembled. In this study, we assessed the effects of several signal peptides, both Sec dependent and Tat dependent, on the translocation of the carboxylesterase in order to optimize the phage display of this enzyme normally restricted to the cytoplasm. Functional display of Bacillus carboxylesterase NA could be achieved when Sec-dependent signal peptides were used. Although a Tat-dependent signal peptide could direct carboxylesterase translocation across the inner membrane of E. coli, proper assembly into phage particles did not seem to occur.In the past decade, the most remarkable successes of protein engineering have been the result of combining random mutagenesis and screening by means of a high-throughput assay (6). Unfortunately, for many enzymes, such as esterases and lipases, no high-throughput methods are available; consequently, evaluation of the enantioselectivity of lipases and esterases is dependent on time-consuming assays (7). Thus, it would be highly advantageous if the screening process could be combined with a rapid selection method that limits the amounts of mutants to be assayed. Phage display (30) is a well-defined technique that has led to a breakthrough in selection methodology for enzymes with desirable properties from a pool of mutants.Derivatives of M13 filamentous phages that are phage particles with a single-stranded genome encapsulated by the phage coat proteins (30) are most commonly used for display in Escherichia coli. Enzymes can be expressed as fusions to one of the M13 phage coat proteins, such as the g3p protein (30). As phage particles are assembled in the cell envelope of E. coli, translocation of the g3p fusion protein across the inner membrane of E. coli is a prerequisite for proper phage display. The g3p protein is synthesized with an 18-residue amino-terminal signal peptide that targets this protein to the E. coli Sec machinery for membrane insertion (26). Theoretically, any protein fused to the amino-terminal region of the g3p protein that is efficiently translocated across the inner membrane and that is able to enter the phage assembly site can be presented as a fusion protein on M13 phages (30).Today, phage display can be used for selection of s...

The twin-arginine transport system: moving folded proteins across membranes

“…Fisher et al [159] addressed this very issue and, using an ingenious tripartite fusion with a Tat signal at the N-terminus, a β-lactamase at the C-terminus and the protein of interest sandwiched in the middle, were able to actively select for variant fusion proteins with increased water solubility. The Tat system has also been exploited in several other protein-engineering projects, including a novel two-hybrid system for screening for protein-protein interactions [160], production and engineering of single-chain antibodies [64,161] and for phage display [162,163]. Some bacteria, in particular Gram-positive Streptomyces spp., export large numbers of diverse substrates by the Tat system and might be ideal hosts for the heterologous production of pharmaceutically important proteins that are incompatible with secretion by the Sec pathway [17].…”

PvdP Is a Tyrosinase That Drives Maturation of the Pyoverdine Chromophore in Pseudomonas aeruginosa

“…For protein localization assays, pvdP_H6 was PCR amplified from pET26B-pvdP_H6 and cloned in the shuttle vector pME6032 (17), obtaining the plasmid pME-pvdP_H6. This plasmid was then transformed in the E. coli HB2151 wild type (WT) and the E. coli HB2151 ⌬tatC mutant (18).…”

“…This plasmid was electroporated into the E. coli HB2151 WT and the E. coli HB2151 ⌬tatC mutant (18). The two strains were grown overnight in 2ϫ TY of LB medium at 37°C and 250 rpm.…”

“…Analyst 1.5.1 software was used for data acquisition and evaluation. Each sample containing approximately 1 mg/ml (suspended in 100% methanol) was diluted 20 times in methanol before injection, and 30 l was injected on a C 18 at 0.6 ml/min, and an additional postcolumn flow was added, providing a flow of 0.2 ml/min of solvent D (consisting of 99.9% acetonitrile and 0.1% formic acid) to improve spray conditions. Elution was performed using a 25-min linear gradient from 5% to 90% solvent B.…”

Heterogeneous catalysis on the phage surface: Display of active human enteropeptidase

“…[27,40,41]. The examples of enzymes that have been phage-displayed to study their action mechanisms include trypsin [42], b-lactamases [43], metallo-b-lactamase b LII from Bacillus cereus [44], amylases [45], subtilisin [46,47], and carboxylesterase of Bacillus subtilis [48]. The mutant variant of 4 0 -phosphopantetheinyl transferase showing a more than 300-fold increase in catalytic efficiency toward 3 0 -dephospho-CoA was selected by co-displaying peptide substrates and enzyme mutants on the M13 phage surface as fusions to the phage capsid protein pIII [49].…”