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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

Western blot analysis of periplasmic extracts directly before, ½ h, 1 h, 2 h, 4 h and 10 h after helper phage superinfection, and of soluble wtPhoA (left lane). (A) Fos-PhoA detected in XL1-Blue harbouring pJuFoIII::phoA without (-) and in the presence of 1 mM IPTG (+). (B) Fos-PhoA detected in XL1-Blue harbouring pJuFoVIII::phoA grown without (-) and in the presence of 1 mM IPTG (+). (C) Growth rate of XL1-blue/pJuFoIII::phoA (squares) and XL1-blue/pJuFoVIII::phoA (circles) grown without (open symbols) and with 1 mM IPTG (solid symbols)
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Figure 3: Western blot analysis of periplasmic extracts directly before, ½ h, 1 h, 2 h, 4 h and 10 h after helper phage superinfection, and of soluble wtPhoA (left lane). (A) Fos-PhoA detected in XL1-Blue harbouring pJuFoIII::phoA without (-) and in the presence of 1 mM IPTG (+). (B) Fos-PhoA detected in XL1-Blue harbouring pJuFoVIII::phoA grown without (-) and in the presence of 1 mM IPTG (+). (C) Growth rate of XL1-blue/pJuFoIII::phoA (squares) and XL1-blue/pJuFoVIII::phoA (circles) grown without (open symbols) and with 1 mM IPTG (solid symbols)

Mentions: 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).


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)

Western blot analysis of periplasmic extracts directly before, ½ h, 1 h, 2 h, 4 h and 10 h after helper phage superinfection, and of soluble wtPhoA (left lane). (A) Fos-PhoA detected in XL1-Blue harbouring pJuFoIII::phoA without (-) and in the presence of 1 mM IPTG (+). (B) Fos-PhoA detected in XL1-Blue harbouring pJuFoVIII::phoA grown without (-) and in the presence of 1 mM IPTG (+). (C) Growth rate of XL1-blue/pJuFoIII::phoA (squares) and XL1-blue/pJuFoVIII::phoA (circles) grown without (open symbols) and with 1 mM IPTG (solid symbols)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Western blot analysis of periplasmic extracts directly before, ½ h, 1 h, 2 h, 4 h and 10 h after helper phage superinfection, and of soluble wtPhoA (left lane). (A) Fos-PhoA detected in XL1-Blue harbouring pJuFoIII::phoA without (-) and in the presence of 1 mM IPTG (+). (B) Fos-PhoA detected in XL1-Blue harbouring pJuFoVIII::phoA grown without (-) and in the presence of 1 mM IPTG (+). (C) Growth rate of XL1-blue/pJuFoIII::phoA (squares) and XL1-blue/pJuFoVIII::phoA (circles) grown without (open symbols) and with 1 mM IPTG (solid symbols)
Mentions: 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).

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