Transgenic plants were screened for presence of the
recombinant protein in leaf tissue and Lf expression
levels were determined by indirect ELISA method. The
amount of plant produced human Lf was calculated by
comparison of the absorbance values obtained for extracts Lf from human milk with a known protein concentration.
Quantitative analysis revealed that human Lf content was
0.0047 % of total soluble protein (TSP) in the clone 53.
Transgenic clone 35, in which human Lf transcripts were
not detected by RT-PCR, expressed the recombinant
protein but in lower level 0.0035 % of TSP. In the other
clones tested, the recombinant protein was not detected.
To confirm expression and the size of the recombinant
human Lf, Western blot analysis was performed. The
protein detected in alfalfa leaf tissue from the clone 53
had a molecular mass of approximately 80 kDa, similar to
that of the full-length native human Lf. A band of the
same size was also detected in leaf extracts from the
clone 35 but the signal was too weak (not shown).
Extracts of non-transformed alfalfa plants did not react
with the anti-human Lf antibody.