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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

Genetic elements of the pJuFo vector and proposed mechanism for the assembly of PhoA-phagemids. In the present work PhoA was displayed either as pIII or pVIII fusion protein via the heterodimeric Jun-Fos linker on the surface of filamentous phagemid
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Figure 1: Genetic elements of the pJuFo vector and proposed mechanism for the assembly of PhoA-phagemids. In the present work PhoA was displayed either as pIII or pVIII fusion protein via the heterodimeric Jun-Fos linker on the surface of filamentous phagemid

Mentions: Among the filamentous phage display approaches the pJuFo technology, which is based on an indirect fusion approach to display products on the surface of helper phage assisted phagemid assemblies, has widely been used for the surface expression of cDNA libraries, and yielded complex allergen repertoires of various allergenic sources [18]. Furthermore, the technology provided a valuable tool for the identification of tumor-associated antigens [19], self-antigens [20] or epitopes of polyclonal antibody sera [21]. Since the applications of this methodology are quite diverse, and yet it has not met substantial limitations, we investigated the efficiency of the pJuFo surface presentation choosing E. coli alkaline phosphatase (PhoA, EC 3.1.3.1) as a model system. PhoA was cloned adjacent to fos of the pJuFo vector and expressed as a Jun-Fos-pIII or Jun-Fos-pVIII fusion protein on the phagemids' surface (Fig. 1). Under reaction conditions in which PhoA exhibits Michaelis-Menten behaviour we obtained the kinetic parameters Km and kcat of phagemid-displayed PhoA. Comparison of these kinetic parameters with those of wild-type PhoA allowed calculating the decoration of phagemid with functional PhoA fusion proteins.


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)

Genetic elements of the pJuFo vector and proposed mechanism for the assembly of PhoA-phagemids. In the present work PhoA was displayed either as pIII or pVIII fusion protein via the heterodimeric Jun-Fos linker on the surface of filamentous phagemid
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Genetic elements of the pJuFo vector and proposed mechanism for the assembly of PhoA-phagemids. In the present work PhoA was displayed either as pIII or pVIII fusion protein via the heterodimeric Jun-Fos linker on the surface of filamentous phagemid
Mentions: Among the filamentous phage display approaches the pJuFo technology, which is based on an indirect fusion approach to display products on the surface of helper phage assisted phagemid assemblies, has widely been used for the surface expression of cDNA libraries, and yielded complex allergen repertoires of various allergenic sources [18]. Furthermore, the technology provided a valuable tool for the identification of tumor-associated antigens [19], self-antigens [20] or epitopes of polyclonal antibody sera [21]. Since the applications of this methodology are quite diverse, and yet it has not met substantial limitations, we investigated the efficiency of the pJuFo surface presentation choosing E. coli alkaline phosphatase (PhoA, EC 3.1.3.1) as a model system. PhoA was cloned adjacent to fos of the pJuFo vector and expressed as a Jun-Fos-pIII or Jun-Fos-pVIII fusion protein on the phagemids' surface (Fig. 1). Under reaction conditions in which PhoA exhibits Michaelis-Menten behaviour we obtained the kinetic parameters Km and kcat of phagemid-displayed PhoA. Comparison of these kinetic parameters with those of wild-type PhoA allowed calculating the decoration of phagemid with functional PhoA fusion proteins.

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