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

Weichel M, Jaussi R, Rhyner C, Crameri R - Open Biochem J (2008)

Bottom Line: The phoA gene was cloned downstream of fos while jun was inserted upstream of pIII or pVIII, alternatively, in the pJuFo phagemid vector.PhoA displayed on the phagemid pIII surface exhibited a K(m) of 11.2 microM with 4-nitrophenyl phosphate as substrate, which is consistent with data published for soluble PhoA.However, since PhoA is displayed in a native-like fashion, as deduced from the kinetic parameters of the enzymatic reaction, the pJuFo technology provides a versatile tool for the functional screening of complex cDNA libraries displayed on the phagemids' surface.

View Article: PubMed Central - PubMed

Affiliation: Swiss Institute of Allergy and Asthma Research (SIAF), Obere Strasse 22, CH-7270 Davos, Switzerland.

ABSTRACT
Active alkaline phosphatase of Escherichia coli (PhoA, EC 3.1.3.1) was displayed via the leucine zipper element of the Jun-Fos heterodimer on the surface of filamentous phage and the kinetic parameters K(m) and k(cat) were determined. The phoA gene was cloned downstream of fos while jun was inserted upstream of pIII or pVIII, alternatively, in the pJuFo phagemid vector. Both fusion genes are regulated by independent lacZ promoters. PhoA displayed on the phagemid pIII surface exhibited a K(m) of 11.2 microM with 4-nitrophenyl phosphate as substrate, which is consistent with data published for soluble PhoA. Based on these data we calculated the decoration of pJuFo phagemid with PhoA using the minor and major coat proteins pIII and pVIII as fusion partners under variable inducing conditions. We found that, even if the promoters are fully induced at a concentration of 1000 microM IPTG, the phagemids display maximally one copy of PhoA-Fos-Jun-coat protein fusion, irrespective of whether the protein is presented via pIII or pVIII. However, since PhoA is displayed in a native-like fashion, as deduced from the kinetic parameters of the enzymatic reaction, the pJuFo technology provides a versatile tool for the functional screening of complex cDNA libraries displayed on the phagemids' surface.

No MeSH data available.


Related in: MedlinePlus

Molar activities of soluble dimeric PhoA (black), PhoA-pIII-phagemid (light grey) and PhoA-pVIII-phagemid (dark grey) in dependence on the IPTG concentration. Molar activities of PhoA-phagemid are measured per phage particle. As control pIII-pJuFo and pVIII-pJuFo phagemids without a PhoA fusion were used. The molar activities of these control samples were 1.33 and 1.99 mol substrate converted/mol phage/min respectively (small figure top left)
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Figure 4: Molar activities of soluble dimeric PhoA (black), PhoA-pIII-phagemid (light grey) and PhoA-pVIII-phagemid (dark grey) in dependence on the IPTG concentration. Molar activities of PhoA-phagemid are measured per phage particle. As control pIII-pJuFo and pVIII-pJuFo phagemids without a PhoA fusion were used. The molar activities of these control samples were 1.33 and 1.99 mol substrate converted/mol phage/min respectively (small figure top left)

Mentions: 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].


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

Weichel M, Jaussi R, Rhyner C, Crameri R - Open Biochem J (2008)

Molar activities of soluble dimeric PhoA (black), PhoA-pIII-phagemid (light grey) and PhoA-pVIII-phagemid (dark grey) in dependence on the IPTG concentration. Molar activities of PhoA-phagemid are measured per phage particle. As control pIII-pJuFo and pVIII-pJuFo phagemids without a PhoA fusion were used. The molar activities of these control samples were 1.33 and 1.99 mol substrate converted/mol phage/min respectively (small figure top left)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2570559&req=5

Figure 4: Molar activities of soluble dimeric PhoA (black), PhoA-pIII-phagemid (light grey) and PhoA-pVIII-phagemid (dark grey) in dependence on the IPTG concentration. Molar activities of PhoA-phagemid are measured per phage particle. As control pIII-pJuFo and pVIII-pJuFo phagemids without a PhoA fusion were used. The molar activities of these control samples were 1.33 and 1.99 mol substrate converted/mol phage/min respectively (small figure top left)
Mentions: 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].

Bottom Line: The phoA gene was cloned downstream of fos while jun was inserted upstream of pIII or pVIII, alternatively, in the pJuFo phagemid vector.PhoA displayed on the phagemid pIII surface exhibited a K(m) of 11.2 microM with 4-nitrophenyl phosphate as substrate, which is consistent with data published for soluble PhoA.However, since PhoA is displayed in a native-like fashion, as deduced from the kinetic parameters of the enzymatic reaction, the pJuFo technology provides a versatile tool for the functional screening of complex cDNA libraries displayed on the phagemids' surface.

View Article: PubMed Central - PubMed

Affiliation: Swiss Institute of Allergy and Asthma Research (SIAF), Obere Strasse 22, CH-7270 Davos, Switzerland.

ABSTRACT
Active alkaline phosphatase of Escherichia coli (PhoA, EC 3.1.3.1) was displayed via the leucine zipper element of the Jun-Fos heterodimer on the surface of filamentous phage and the kinetic parameters K(m) and k(cat) were determined. The phoA gene was cloned downstream of fos while jun was inserted upstream of pIII or pVIII, alternatively, in the pJuFo phagemid vector. Both fusion genes are regulated by independent lacZ promoters. PhoA displayed on the phagemid pIII surface exhibited a K(m) of 11.2 microM with 4-nitrophenyl phosphate as substrate, which is consistent with data published for soluble PhoA. Based on these data we calculated the decoration of pJuFo phagemid with PhoA using the minor and major coat proteins pIII and pVIII as fusion partners under variable inducing conditions. We found that, even if the promoters are fully induced at a concentration of 1000 microM IPTG, the phagemids display maximally one copy of PhoA-Fos-Jun-coat protein fusion, irrespective of whether the protein is presented via pIII or pVIII. However, since PhoA is displayed in a native-like fashion, as deduced from the kinetic parameters of the enzymatic reaction, the pJuFo technology provides a versatile tool for the functional screening of complex cDNA libraries displayed on the phagemids' surface.

No MeSH data available.


Related in: MedlinePlus