Expression of plant genes in transfected mammalian cells: accumulation of recombinant preLHCIIb proteins within cytoplasmic inclusion bodies.
Transfection of the monkey COS-7 cells with an expression vector bearing the Lemma gibba LHCIIb AB30 or AB19 genes led to the synthesis of the LHCIIb polypeptide precursors (preLHCIIb). This was inferred mainly from Western blot analysis which has revealed the appearance of a single immunoprecipitation band following the use of three different preparations of anti-LHCIIb antibodies. Synthesis of the precursor polypeptides, not the mature processed LHCIIb protein, was evident from the molecular weight of the newly synthesized protein, inferred from its position in the gel. Expression of the AB30 and AB19 genes was also evident from the appearance of specific transcripts only in transfected cells. Immunofluorescence observations revealed the appearance of distinct fluorescent spots in about 1-2% of the transfected cells. The same was observed following immunogold staining and electron microscopy studies, which revealed a specific association of gold particles with amorphous structures only in transfected cells. The preLHCIIb synthesized by transfected COS-7 cells was insoluble in a variety of detergents and could be solubilized only by 8 M urea or 0.1 N NaOH. These properties are characteristic of proteins accumulating within inclusion bodies of prokaryotes.
Broido
S
S
Department of Biological Chemistry, Hebrew University of Jerusalem, Israel.
Loyter
A
A
Vainstein
A
A
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D015870:Gene Expression / Q000502:physiology; D045322:Photosynthetic Reaction Center Complex Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D010944:Plants / Q000235:genetics*; D011498:Protein Precursors / Q000096:biosynthesis; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis; D012995:Solubility; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection
D045322:Photosynthetic Reaction Center Complex Proteins; D011498:Protein Precursors; D011994:Recombinant Proteins