Prokaryotic Expression and Allergenicity Assessment of Hygromycin B Phosphotransferase Protein Derived from Genetically Modified Plants

Abstract

The hygromycin B phosphotransferase gene (hpt) has been widely used in the process of plant genetic engineering to produce plants that can secrete the HPT protein. As part of a safety assessment, sufficient quantities of the protein were produced in Escherichia coli to conduct in vitro digestibility and animal studies. Western blotting analysis showed that the HPT protein was digested by simulated gastric fluid within 40 s. ELISA demonstrated that the protein did not induce detectable levels of specific IgE antibodies or histamine in test animals. Alignment of the amino acid sequence of HPT with those of known allergens did not produce evidence of sequence similarities between these allergens and the HPT protein. We conclude that HPT has a low probability to induce allergenicity.

Safety assessment of nonbrowning potatoes: opening the discussion about the relevance of substantial equivalence on next generation biotech crops

“…Because a hairpin silencing strategy was used and no novel protein, other than the already proven safe hygromycin phosphotransferase (HPT) (Petersen et al. , 2005; Lu et al. , 2007; Zhuo et al.…”

“…Furthermore, neither toxicity nor allergenicity is expected to occur from the Hpt selector gene product, because its safety has been previously established (Petersen et al. , 2005; Lu et al. , 2007; Zhuo et al.…”

“…Mice fed -PPO tubers consume more potato than mice fed WT tubers and present normal physiological parameters To investigate whether the reduced enzymatic browning levels in the -PPO potato tubers produce a particular effect on palatability and diet, and considering that an influence of (and on) food intake could happen as a result of the differences originated by the genetic modification (browning inhibition and metabolite levels), feeding studies were conducted in mice. Because a hairpin silencing strategy was used and no novel protein, other than the already proven safe hygromycin phosphotransferase (HPT) (Petersen et al, 2005;Lu et al, 2007;Zhuo et al, 2009), was introduced in the -PPO potatoes, a whole food feeding approach was decided. Animals were fed diets consisting of pellet food supplemented with WT or -PPO potato tubers (both pellet food and potato lines were available ad libitum).…”

Expression, purification and refolding of recombinant Cry1Ab/Ac obtained in Escherichia coli as inclusion bodies

“…The inclusion bodies protein, the purified Cry1Ab/Ac fusion protein and the Cry1Ab/Ac protein without His‐tag were run on 100 g L −1 SDS‐PAGE and transferred onto a cellulose acetate film (Millipore, USA). The film was blocked in 50 g L −1 skimmed milk for 2 h at room temperature, then incubated with Anti‐Cry1Ab/Ac rabbit polyclonal antibody (or His‐tag monoclonal antibody) for 2 h, washed with TBS (TBS) six times, further incubated with alkaline phosphatase‐conjugated goat anti‐rabbit IgG secondary antibody (or alkaline phosphatase‐conjugated goat anti‐mouse IgG secondary antibody) for 2 h, washed again with TBS six times and finally developed with BCIP/NBT (BCIP/NBT) solution 13…”

Transgenic Tall Fescue and Maize with Resistance to ALS-Inhibiting Herbicides

“…The most widely used selectable markers are antibiotic-resistant genes such as the hpt gene and the nptII gene encoding neomycin phosphotransferase. HPT has a low likelihood of inducing toxicity and allergenicity (Zhuo et al, 2009;Lu et al, 2007), and NPTII was determined to be nontoxic for human or animal consumption (Nap et al, 1992). Herbicideresistant genes are also used as selectable markers.…”