The 5-enolpyruvylshikimate-3-phosphate synthase of glyphosate-tolerant soybean expressed in Escherichia coli shows no severe allergenicity.
The recombinant gene was amplified from the chromosomal DNA of genetically-modified (GM) soybeans and identified as epsps encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) which renders glyphosate resistance. The epsps structural gene was introduced in the pET28(a) plasmid for its expression in Escherichia coli BL21(DE3). It was confirmed that the maximal productivity of the EPSPS protein was achieved when cultivating the recombinant strain in a LB broth for 2 h after supplementing 1 mM isopropylbeta-D-thiogalactopyranoside (IPTG) in a 2 h-culture broth. Since the expressed EPSPS protein was found as an insoluble form in the inclusion body, it was extracted by 6 M urea after sonication, and then purified through immobilized nickel-affinity column chromatography to isolate EPSPS having a molecular mass of 57 kDa. When incubated in simulated gastric fluid containing pepsin at pH 1.5, the purified EPSPS protein was completely digested within 1 min. In addition, the passive cutaneous anaphylaxis reaction of the purified EPSPS protein was not observed in the Sprague Dawley rat system that was administered either orally or subcutaneously. Furthermore, treatment of the EPSPS protein to the culture of the sensitized peritoneal mast cells, or unsensitized but antisera-labeled mast cells, showed neither a remarkable change in the histamine release nor a cytokine production, including interleukin-4 (IL-4) and tumor necrosis factor-alpha (TNF-alpha). Thus, it can be concluded that the EPSPS protein in the GM soybean showed no significant allergenicity in the Sprague Dawley rats.
Chang
Hyun Sung
HS
College of Pharmacy, Yeungnam University, Gyongsan 712-749, Korea.
Kim
Nam Hee
NH
Park
Myong Jin
MJ
Lim
Si Kyu
SK
Kim
Sung Chull
SC
Kim
Ji Young
JY
Kim
Jung Ae
JA
Oh
Hye Young
HY
Lee
Chu Hee
CH
Huh
Keun
K
Jeong
Tae Cheon
TC
Nam
Doo Hyun
DH
D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D000284:Administration, Oral; D019883:Alkyl and Aryl Transferases / Q000009:adverse effects* / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology; D000485:Allergens / Q000009:adverse effects* / Q000235:genetics / Q000276:immunology; D000818:Animals; D004044:Dietary Proteins / Q000009:adverse effects* / Q000276:immunology; D004351:Drug Resistance; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli; D005512:Food Hypersensitivity / Q000209:etiology* / Q000517:prevention & control; D030841:Food, Genetically Modified / Q000009:adverse effects*; D005750:Gastric Juice / Q000378:metabolism; D005998:Glycine / Q000031:analogs & derivatives* / Q000494:pharmacology; D006540:Herbicides / Q000494:pharmacology; D006636:Histamine Release / Q000187:drug effects; D007279:Injections, Subcutaneous; D015847:Interleukin-4 / Q000378:metabolism; D008297:Male; D008407:Mast Cells / Q000187:drug effects / Q000378:metabolism; D010323:Passive Cutaneous Anaphylaxis; D010529:Peritoneal Cavity / Q000166:cytology; D010940:Plant Proteins / Q000009:adverse effects* / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology; D010957:Plasmids / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D013025:Glycine max / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D013047:Specific Pathogen-Free Organisms; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D000097797:Glyphosate
D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases; D000485:Allergens; D004044:Dietary Proteins; D004791:Enzyme Inhibitors; D005998:Glycine; D006540:Herbicides; D015847:Interleukin-4; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D000097797:Glyphosate