A stable phage-display system using a phagemid vector: phage display of hen egg-white lysozyme (HEL), Escherichia coli alkaline, phosphatase, and anti-HEL monoclonal antibody, HyHEL10.
A stable expression system for displaying the pIII fusion protein on the surface of a filamentous phage was constructed. A phagemid pIII display vector, pLUCK, was constructed by inserting the gene encoding the pIII fusion protein in the opposite direction to that of the lac promoter of pTZ18U. Using this phage display system, two enzymes, hen egg-white lysozyme (HEL) and E. coli alkaline phosphatase, and the single-chain Fv fragment of anti-HEL monoclonal antibody HyHEL10, could be stably and functionally displayed. Northern and primer extension analyses showed that a small amount of the sense mRNA encoding pIII-fused HEL was transcribed from the minor phage promoter located in the region encoding the C-terminus of pIII. Repressed expression of the pIII fusion protein can lead to the display of a wide range of proteins on filamentous phages without the need for strict expression conditions.
Maenaka
K
K
Department of Biochemistry and Engineering, Graduate School of Engineering, Tohoku University, Sendai, Japan.
Furuta
M
M
Tsumoto
K
K
Watanabe
K
K
Ueda
Y
Y
Kumagai
I
I
D000469:Alkaline Phosphatase / Q000737:chemistry*; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D017104:Bacteriophage M13 / Q000235:genetics; D001483:Base Sequence; D002213:Capsid / Q000737:chemistry; D036022:Capsid Proteins; D003090:Coliphages / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry*; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D014760:Viral Fusion Proteins / Q000737:chemistry
D000911:Antibodies, Monoclonal; D036022:Capsid Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014760:Viral Fusion Proteins; D000469:Alkaline Phosphatase; D009113:Muramidase