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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

Phage yields (solid bars), and infectivities (diagonals) of PhoA-pIII-phagemid (light grey) and PhoA-pVIII-phagemid (dark grey) prepa-rations in the absence and presence of IPTG during phage production. 100% represent 4·1013 phagemids/ml (PhoA-pIII-phagemid) and 9·1013 phagemids/ml (PhoA-pIII-phagemid) respectively. Infectivity is measured in transforming units per ml.
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Figure 6: Phage yields (solid bars), and infectivities (diagonals) of PhoA-pIII-phagemid (light grey) and PhoA-pVIII-phagemid (dark grey) prepa-rations in the absence and presence of IPTG during phage production. 100% represent 4·1013 phagemids/ml (PhoA-pIII-phagemid) and 9·1013 phagemids/ml (PhoA-pIII-phagemid) respectively. Infectivity is measured in transforming units per ml.

Mentions: 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).


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)

Phage yields (solid bars), and infectivities (diagonals) of PhoA-pIII-phagemid (light grey) and PhoA-pVIII-phagemid (dark grey) prepa-rations in the absence and presence of IPTG during phage production. 100% represent 4·1013 phagemids/ml (PhoA-pIII-phagemid) and 9·1013 phagemids/ml (PhoA-pIII-phagemid) respectively. Infectivity is measured in transforming units per ml.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Phage yields (solid bars), and infectivities (diagonals) of PhoA-pIII-phagemid (light grey) and PhoA-pVIII-phagemid (dark grey) prepa-rations in the absence and presence of IPTG during phage production. 100% represent 4·1013 phagemids/ml (PhoA-pIII-phagemid) and 9·1013 phagemids/ml (PhoA-pIII-phagemid) respectively. Infectivity is measured in transforming units per ml.
Mentions: 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).

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