Figure 1
Surface display of the EstA passenger domain. (A) Structural model of P. stutzeri A15 EstA (from D24 to L636) constructed using I-TASSER [23]. (B) Schematic representation of proteins expressed from pHERD26T-estA (EstA), pEstAβ/pEstAβL and pEstA/pEstA*. (A B) Residues delineating the part of EstA used for constructing pEstAβ/pEstAβL (EstAβ) are in blue (A311 and L636). Residues marking the esterase passenger domain (EstAP) are coloured in magenta (P27 and S325). Residues for generating the full-length AT based constructs pEstA/pEstA* (V20, black and L636, blue; see further) have been depicted. The difference between pEstA and pEstA*, mutagenesis of the catalytic serine residue (S*A), has been indicated. The GDSL esterase passenger domain (GDSL), the α-helical linker domain (α) and the β-barrel domain have been depicted in red, yellow and orange respectively, signal peptide (SP), E-epitope tag (E), SfiI restriction site (SfiI). (C) Induced cells of P. stutzeri A15 pHERD26T-estA, pEstAβestAP, pEstAβL-estAP or empty vector (EV) were treated with proteinase K (PK +) or mock-treated (PK -) and analyzed with Western blot using anti-EstA serum (α-EstA) or anti-GroEL (α-GroEL). (D-E) Relative esterase activity of (D) cells of P. stutzeri A15 pHERD26T-estA, pEstAβ-estAP, pEstAβL- estAP or empty vector (EV) or (E) membrane fractions containing equal amounts of EstA/EstAP (Additional file 1: Figure S1). Data represent the mean of three independent repeats ± 95% confidence interval. The significance level (P < 0.05) as determined by one-way ANOVA with Student-Newman-Keuls post-hoc analysis, is indicated with a letter code. Molecular weight markers (kDa) are indicated at the side.