Hyperexpression of chicken riboflavin carrier protein: antibodies to the recombinant protein curtail pregnancy in rodents.
Chicken riboflavin carrier protein (RCP) is a phosphoglycoprotein present in the egg white and yolk of egg-laying animals and in the sera of laying hens and of estrogenized chicks. The RCP cDNA, encoding a protein of predicted M(r) 27,000, has been cloned into a T7 polymerase-driven vector, and high-level expression was observed on induction with IPTG in Escherichia coli. The protein was largely localized in inclusion bodies when expressed at 37 degrees C but was present in the cytosolic fraction when induced at 22 degrees C. At 37 degrees C, two major bands were detected in whole-cell lysates of the strain expressing the protein. N-terminal sequence analysis indicated that the two proteins represented translated products with and without the pelB leader sequence encoded in the pET20b vector, but both included an additional 10 amino acids generated during cloning procedures. The inclusion body obtained at 37 degrees C, on extraction with detergent, led to preferential solubilization of the protein without the pelB signal sequence. The solubilized recombinant RCP was recognized by polyclonal antisera to native RCP but radioimmunoassay revealed quantitative differences in the epitopes exhibited by the recombinant protein. Thus, sequence-specific monoclonal antibodies to chicken RCP also cross-reacted with the recombinant protein with almost equal efficiency, but antibodies which recognize conformation-dependent epitopes showed relatively reduced cross-reactivity with the recombinant protein. Polyclonal antibodies to recombinant RCP were able to recognize both the native and the denatured RCP. Administration of recombinant RCP antisera to pregnant mice led to embryonic resorption leading to early pregnancy termination. These findings reveal that the recombinant protein will be useful for investigations related to the mechanism of pregnancy termination on immunoneutralization of RCP in mammals, as well as in unraveling folding properties of RCP in terms of its ligand binding and antigenetic determinants exposed at its surface.
Sooryanarayana
Department of Biochemistry, Indian Institute of Science, Bangalore, India.
Adiga
P R
PR
Visweswariah
S S
SS
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D001483:Base Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000276:immunology*; D002645:Chickens / Q000378:metabolism; D003267:Contraception; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D005298:Fertility / Q000187:drug effects; D005786:Gene Expression Regulation / Q000235:genetics; D007114:Immunization; D026901:Membrane Transport Proteins; D051379:Mice; D008969:Molecular Sequence Data; D011247:Pregnancy; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification*; D012256:Riboflavin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D017931:DNA Primers; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; C019079:riboflavin-binding protein; D012256:Riboflavin