Cloning of the J gene of bacteriophage lambda, expression and solubilization of the J protein: first in vitro studies on the interactions between J and LamB, its cell surface receptor.
Bacteriophage lambda adsorbs to its Escherichia coli K12 host by interacting with a specific cell surface receptor, the outer membrane protein LamB. Previous genetic analyses led us to define a set of residues at the surface of LamB, which belong to the lambda receptor site. Further genetic studies indicated that the C-terminal portion of J, the tail fibre protein of lambda, was directly involved in the recognition of the receptor site. The present work describe first in vitro studies on the interactions between J and LamB. The J gene of lambda was cloned into a plasmid vector under ptac promoter control and expressed in E. coli. We showed that J could be expressed at high levels (up to 28% of whole cell proteins), in an insoluble form. Anti-J antibodies, induced in rabbits immunized with insoluble J extracts, appeared to specifically neutralize lambda infection. Under defined conditions of extraction, the J protein was obtained in a soluble form. We showed that solubilized J was able to interact with LamB trimers in vitro. Implications for future studies on the interactions between LamB and J are discussed.
Wang
J
J
Laboratory of Molecular Genetics, Institute of Biotechnology, Fengtai District, Beijing, P.R. China.
Michel
V
V
Hofnung
M
M
Charbit
A
A
D000818:Animals; D000914:Antibodies, Viral; D001425:Bacterial Outer Membrane Proteins; D010582:Bacteriophage lambda / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D015153:Blotting, Western; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism / Q000821:virology; D005822:Genetic Vectors; D015151:Immunoblotting; D009500:Neutralization Tests; D010957:Plasmids / Q000235:genetics; D018272:Porins; D011817:Rabbits; D011991:Receptors, Virus / Q000737:chemistry / Q000378:metabolism*; D012995:Solubility; D017376:Viral Tail Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*
D000914:Antibodies, Viral; D001425:Bacterial Outer Membrane Proteins; C083033:J protein, Bacteriophage lambda; D018272:Porins; D011991:Receptors, Virus; D017376:Viral Tail Proteins; C016532:maltoporins