Expression of a recombinant human sperm-agglutinating mini-antibody in tobacco (Nicotiana tabacum L.).
The murine monoclonal antibody (mAB) S19 recognizes an N-linked carbohydrate antigen designated sperm agglutination antigen-1 (SAGA1) located on the membrane protein CD52. This antigen is added to the sperm surface during epididymal maturation. Binding of the S19 mAB to SAGA-1 causes the rapid agglutination of sperm and blocks pre-fertilization events. Previous studies indicated that the S19 mAB may be a potential specific spermicidal agent (termed a spermistatic) capable of replacing current spermicidal products that contain harsh detergents with harmful side effects. The nucleotide sequences encoding the heavy (H) and light (L) chains of the S19 antibody were cloned. A chimeric gene was constructed using the nucleotide sequences encoding the variable regions of both the H and L chains, and this gene (scFv1 9) was expressed in transgenic tobacco (Nicotiana tabacum L.) to produce a recombinant anti-sperm antibody (RASA). Highest levels of RASA expression were observed in BY-2 plant cell suspension cultures and regenerated N. tabacum cv. Xanthi plants transformant in which the RASA coding sequences were expressed under the control of the Cauliflower Mosaic Virus 35S promoter containing a double-enhancer sequence (2X CaMV 35S). Subsequent modifications of the transgene including the addition of a 5'-untranslated sequence from the tobacco etch virus (TEV leader sequence), N-terminal fusion of the coding region with an endoplasmic reticulum targeting signal of patatin (pat) and C-terminal fusion with the endoplasmic reticulum retention signal peptide KDEL showed further enhancement of RASA expression. The plant-expressed RASA formed intrachain disulfide bonds and was primarily soluble in the cytoplasmic fraction of the cells. Introduction of a poly-histidine (6xHIS) tag in the recombinant RASA protein allowed for rapid purification of the recombinant protein using Ni-NTA chromatography. Optimization of scale-up production and purification of this plant-derived recombinant protein should provide large quantities of an inexpensive spermistatic plantibody.
Xu
Bingfang
B
Departments of Biology, University of Virginia, Charlottesville, Virginia, 22904, USA.
Copolla
Michael
M
Herr
John C
JC
Timko
Michael P
MP
D000911:Antibodies, Monoclonal / Q000302:isolation & purification* / Q000494:pharmacology; D019149:Bioreactors; D002478:Cells, Cultured; D003269:Contraception, Immunologic; D003272:Contraceptive Agents, Male / Q000302:isolation & purification* / Q000494:pharmacology; D003600:Cytosol / Q000276:immunology; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D005818:Genetic Engineering; D006801:Humans; D008297:Male; D030821:Plants, Genetically Modified; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D013073:Sperm Agglutination / Q000276:immunology*; D013094:Spermatozoa / Q000276:immunology; D014026:Nicotiana / Q000235:genetics / Q000276:immunology*; D022642:Vaccines, Contraceptive / Q000302:isolation & purification* / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D003272:Contraceptive Agents, Male; D011994:Recombinant Proteins; D022642:Vaccines, Contraceptive