Display of E. coli Alkaline Phosphatase pIII or pVIII Fusions on Phagemid Surfaces Reveals Monovalent Decoration with Active Molecules

Construction of pJuFoVIII : : phoA

Production of phage displaying PhoA of E. coli as a pIII fusion protein by means of the Jun-Fos linker is described elsewhere [11, 37]. For the construction of a pVIII-display pJuFo vector, the DNA encoding pVIII was amplified by PCR using M13K07 helper phage as a template and the following oligonucleotides: 5^'-primer pVIII: 5^'-ACG GGA TCC GGT GGC GGT GGC TCT GCT GAG GGT GAC-3^', 3^'-primer pVIII: 5^'-GTG TTA CTA GCT AGC TTT AAT TGT ATC GGT TTA-3^'. The PCR fragment was hydrolysed with BamHI and NheI, gel purified and used to replace the BamHI-NheI fragment in pJuFo. The ligation mixture was used to transform E. coli XL1-Blue (Stratagene, La Jolla, CA, USA) electrocompetent cells [38] which were then spread on LB agar plates with 100 µg/ml ampicillin and 10 µg/ml tetracycline (LB/Amp+Tc). Single colonies were picked and grown. DNA was prepared and the final construct was was verified by restriction analysis and DNA sequencing (Microsynth, Balgach, Switzerland). The pJuFoVIII:: phoA phagemid was constructed by XbaI+KpnI hydrolysis of the pJuFoVIII vector and ligation with correspondingly hydrolysed phoA DNA derived from pJuFoIII::phoA [11].

Preparation of PhoA-Phagemids

110-150 ml 2×YT medium with 100 µg/ml ampicillin and 10 µg/ml tetracycline (2×YT/Amp+Tc) were inoculated 1:50 with a stationary culture of E. coli XL1-Blue/pJuFo::phoA or XL1-Blue/pJuFoVIII::phoA and incubated at 37^°C and 250 rpm until an OD₅₅₀ of ≈ 0.5 was reached. Then helper phage VCS-M13 (Stratagene) was added at a multiplicity of infection (moi) of 10 and the culture was incubated for 30 min at 30^°C, 150 rpm. After 30 min agitation of the culture was raised to 300 rpm and the culture was incubated for additional 9.5 h at 30^°C. 2 h after infection with helper phage, kanamycin (kan) was added to a concentration of 70 µg/ml. For experiments, in which the decoration of phage with PhoA was determined in dependence of the inducer concentration, IPTG was added 30 min after infection with helper phage.

PhoA-phagemid containing supernatant was prepared 10 h after helper phage addition by centrifugation (4'500 × g, 4^°C, 15 min) and PhoA-phagemid was precipitated by addition of ¼ volume 25% (w/v) polyethylene glycol (MW: 8'000), 15% (w/v) NaCl at 4^°C for 20 min. After centrifugation (15'000 × g, 4^°C, 20 min) the supernatant was removed and the precipitated phagemid was dissolved in 10 mM Tris/HCl pH 8.0 (4 vol. % of the original supernatant volume). After centrifugation (15'000 × g, 4^°C, 10 min) the cleared solution was transferred into fresh tubes and PhoA-phagemid was precipitated again with PEG/NaCl as described above, the supernatant was removed quantitatively and precipitated PhoA-phagemid was dissolved in 10 mM Tris/HCl pH 8.0 (4 vol. % of the original supernatant volume). After final centrifugation (15'000 × g, 4^°C, 10 min) the PhoA-phagemid solution was transferred into new tubes, kept at 4^°C and readily used for the determination of infectivity and for enzymatic assays. Samples immediately not used for experiments were stored at -20^°C directly after preparation.

Inducibility of pJuFoIII: : phoA and pJuFoVIII : : phoA

To test the inducibility of the phagemid vectors by IPTG after addition of helper phage we prepared periplasmic extracts of XL1-Blue using a moderately modified protocol for cold osmotic shock [39]: portions of 25 ml were taken from a 200 ml PhoA-phagemid producing XL1-Blue culture at distinct times after VCS-M13 superinfection and centrifuged (4'500 × g, 4^°C, 15 min). The supernatant was removed completely and bacterial pellets washed by gentle re-suspension in 1 ml of 2×YT followed by centrifugation (4'500 × g, 4^°C, 15 min). After medium removal the bacteria were resuspended in 1 ml 500 mM Saccharose, 100 mM Tris/HCl, 1 mM EDTA pH 8.0 and incubated on ice for 30 min in the presence of 100 µg/ml hen egg white lysozyme (Roche, Basel, Switzerland). The spheroplasts were sedimented (15'000 × g, 4^°C, 15 min) and the periplasmic fractions were stored at -20^°C.

Western Blots

Western blot analysis was used to detect wild-type (wt) PhoA (Sigma, St. Louis, MO, USA) or Fos-PhoA in the periplasmic fractions as well as in the phagemid preparations. Therefore, samples were subjected to SDS-PAGE (NuPAGE^™, 12% Bis-Tris, Invitrogen^™) and electrotransferred onto a Hybond^™-P PVDF membrane (Amersham Pharmacia Biotech, Uppsala, Sweden). Free binding sites were blocked with 5% non-fat dried milk, 0.1 % (v/v) Tween-20 in TBS. PhoA was detected with horse radish peroxidase labelled rabbit polyclonal anti-bacterial AP mAb 7319 (1:10'000 in blocking buffer, Abcam, Cambridge, United Kingdom) and visualised by chemiluminescence (ECL^™, Amersham Pharmacia Biotech) substrate.

Enzymatic Assays

PhoA-phagemid enzyme activity was assayed basically as described by McCafferty et al. [31]. Briefly, reactions were initiated by adding 50 µl PhoA-phagemid solution to 950 µl of 4-nitrophenyl phosphate (Fluka, Buchs, Switzerland) at a range of concentrations in 1.052 M Tris/HCl pH 8.0. The initial rates were calculated from the change of the absorbance at 410 nm in the first 6 s of each reaction at 25^°C using a molar absorption coefficient of 16'200 M^-1 cm^-1 [40]. The concentration of PhoA-phagemid was determined by preparing ssDNA using phenol/chloroform/ isopropanol extraction, ethanol precipitation and measuring at 260 nm. The A₂₆₀/A₂₈₀ ratio of the ssDNA prepared this way was always higher than 1.77. The molar absorption coefficients of the individual single stranded vector contructs were calculated with the Omiga 2.0 software (Oxford Molecular, Oxford, England). Purified E. coli PhoA was purchased from Sigma. The concentration was calculated by measuring the absorbance at 278 nm [41] using a M_r of 47'029 per subunit [28]. The kinetic parameters K_m and k_cat were derived fromnonlinear least-square fits according to the Michaelis-Menten model. For practical reasons k_cat values are expressed as molar activities (mol. act.) and specified in mol substrate/mol phage (or enzyme)/min throughout this paper. All absorption measurements were performed on an Uvikon XL spectrophotometer (Bio-Tek Instruments, Winooski, VT, USA).

Phagemid Infectivity

Infectivity is defined as the ratio between infective and total phage. Total phagemid was measured by preparing ssDNA as described above, infective phagemid was determined by infecting exponentially growing E. coli XL1-Blue with three independent dilution series of each phagemid preparation and plating out on LB/Amp+Tc agar.

Kinetic Properties of PhoA Displayed on the Surface of pJuFo Phagemids

Display of cDNA libraries on the surface of filamentous phage and their screening for interacting proteins against immobilized targets still remains a challenge in proteomics. Although also pVI as fusion partner for cDNA products has been described [13, 14, 22], the pJuFo technology which uses the pIII-Jun-Fos-cDNA fusion is well established for the efficient selection and identification of protein-protein interacting partners [16]. To determine if these proteins are displayed in a native-like conformation, we have generated phagemid vectors displaying E. coli PhoA on the surface of filamentous phage particles as pIII or pVIII fusion proteins via the Jun-Fos linker, determined the kinetic parameters of the phagemid-displayed enzymes and compared them to those of soluble PhoA. It has been shown previously that functional PhoA can be displayed on the surface of filamentous phage [11, 14, 15, 23]. However, only McCafferty et al. [23] provide detailed kinetic data and show that PhoA directly fused to the N-terminus of pIII exhibits a different K_m compared to soluble PhoA.

As shown in Table 1 PhoA displayed on pJuFo –III phagemids exhibits a K_m of 11.2 µM, which is in agreement with published data of soluble PhoA [23, 24]. This value is independent from PhoA being displayed as pIII or pVIII fusion and independent of the IPTG concentration under which the nascent PhoA-phagemid is being produced.

Assuming that the individual rate constants composing K_m of PhoA-phagemid are comparable to those of soluble PhoA the number of active enzyme molecules displayed on each phagemid particle can be calculated. Fig. (2) shows the molar activities of wtPhoA, PhoA-pIII-phagemid and PhoA-pVIII-phagemid produced in the absence of IPTG. Under this condition PhoA is presented nearly twice as effectively via pIII than via pVIII. However, PhoA-pIII-phagemid shows a molar activity of 1587 mol substrate/mol phage/min compared to a molar activity of 3539 mol substrate/mol enzyme/min for soluble PhoA, indicating that only 45% of the phagemids display one active enzyme molecule via pIII.

Another aim of this study was to investigate if and to what extend addition of IPTG during phagemid production would increase the decoration of phagemids with active enzyme. We therefore tested in a first step the inducibility of the pJuFo: : phoA vectors by preparing periplasmic fractions from phagemid producing XL1-Blue cultures grown with or without 1 mM IPTG. The periplasmic fractions were then subjected to Western blot analysis and the Fos-PhoA fusion protein was detected by HRP-labelled anti-bacterial PhoA mAb 7319. As shown in Fig. (3), induction of fos: : phoA expression is enhanced with IPTG in either of the phagemid vectors during the entire period of phage propagation. Surprisingly, the difference in fos::phoA expression with and without IPTG is much less pronounced in pJuFoIII::phoA (Fig. 3A) compared to pJuFoVIII: : phoA (Fig. 3B). This difference might be more obvious if we consider that the periplasmic extracts of the cultures grown in medium with 1 mM IPTG were prepared from a 10 to 15% reduced cell number compared to those grown without IPTG (Fig. 3C).

To directly investigate the effect of the inducer on the valency of PhoA-phagemids we then added IPTG to final concentrations of 1 µM, 10 µM, 100 µM and 1000 µM 30 min after 10 µM, 100 µM and 1000 µM 30 min after infecting the bacteria with helper phage and prepared the phagemids after another 9½ h. The molar activities of these PhoA-phagemids are shown in Fig. (4). We observed a twofold increase of PhoA-pVIII-phagemid activity in the range between 1 µM and 10 µM IPTG with an apparent activity saturation at IPTG concentrations higher than 10 µM. An increase in molar activity could also be detected in the case of PhoA-pIII-phagemids at IPTG concentrations ≥ 10 µM, but it was much less pronounced. Because both jun: : gIII and fos: : phoA are under control of lacZ promotors, we consider this behaviour to reflect the in vivo dissociation constant of inducer and Lac repressor, which is 5.7 µM [25].

The finding that a maximum of one copy of PhoA is displayed on either species of pJuFo phagemids could also be confirmed by Western blot analysis (Fig. 5).

However, it should be annotated that both total phagemid yield as well as their infectivity decrease with increasing IPTG concentration as shown in Fig. (6).

The titer of PhoA-pIII-phagemids produced at IPTG concentrations ≥ 10 µM was less than 50 % compared to the phagemid titers obtained without induction, while infectivity decreased almost twofold from 1.8 to 1.0 percent. For PhoA-pVIII-phagemids the total yield even dropped below 30 % at IPTG concentrations ≥ 10 µM, while infectivity decreased from 5.5 (no IPTG) to 3.3 percent (1000 µM IPTG) and thus was always higher compared to the pIII fusion. However, infectivity of PhoA-pVIII-phagemids decreased significantly after 48 h storage at 4^°C, indicating that the phage displaying the PhoA-pVIII fusion protein is less stable than the phage displaying the PhoA-pIII fusion protein (data not shown). Therefore we recommend the use of pIII as a fusion partner for phage surface display of cDNA libraries.