Expression of Human Milk Proteins in Microorganisms Microorganisms such as Saccharomyces and Aspergillus are being used for expression of human milk proteins. Human lactoferrin has been expressed in Saccharomyces, but is not commercially available, possibly due to low expression levels . Aspergillus, however, is successfully used for the production of recombinant human lactoferrin , which is commercially available. Expression levels are very high, making it an attractive system; however, extensive purification is needed and the cost is most likely too high for use as a food additive. Consequently, pharmaceutical applications, being more cost tolerant, are being pursued for recombinant proteins produced in this expression system.
Expression of Human Milk Proteins in Milk of Dairy Animals Some of the first commercial applications for recombinant proteins were obtained following their expression in the milk of transgenic animals.
Pharmaceutical recombinant proteins, such as clotting factors VIII and IX, were early expressed in the milk of sheep and goats. Recombinant human lactoferrin, a major protein component of human milk, is being expressed in transgenic cows  and is now commercially available. Purified recombinant human lactoferrin may, however, be too expensive to be used as a food additive or in infant formula. One possible application would be to use cow's milk containing recombinant human milk proteins, such as lactoferrin, but it has not yet found a market, possibly due to logistics (transport, storage) and/or cost. Human lysozyme has been expressed in the milk of mice , and more recently in cows [E. Maga, personal commun.], but, again, there are no commercial applications yet.
Plant biologists have successfully been able to express recombinant proteins in various crops. By using strong promoters, high levels of expression can be achieved. It is also possible to direct the expression so that specific parts of the plant can be utilized, depending on the species. Fruit (bananas), seeds (rice, barley), leaves (tobacco) and tubers (potatoes) are all used for expression of various proteins.
Human lactoferrin has been produced in tobacco . Expression levels were relatively low, however, and the protein needs to be purified extensively before it could be considered for any food applications, making it unlikely as a commercially viable product. There have been no tests of activity of this protein, and it is not yet commercially available. Human lactoferrin has also been expressed in potatoes , but it appears that expression levels so far are low. This system is attractive in that potatoes are a normal part of the diet of many people; however, it is uncertain if the protein will have any biological activity after the extensive boiling that is used for potatoes.
Rice has been used for expression of soybean ferritin to increase its iron content [14, 15] and is now being used for the expression of several human milk proteins, such as lactoferrin, lysozyme and a1-antitrypsin [16-21]. Very high expression levels can be achieved; e.g. 5g of human lactoferrin/kg dehusked rice has been expressed in large scale field trials for several generations . Rice has several advantages as an expression system: (1) rice does not contain any toxic compounds (potatoes contain solanin); (2) rice is one of the first 'non-milk' foods introduced to infants, which in part is due to its low allergenicity, and (3) expression can be directed so the protein is either expressed as a storage protein (in the seed) driven by the Gt1 promoter, or when driven by the amylase promoter, expressed only during germination, making malting another possibility. In this case, the crop (i.e. rice seeds) does not contain the recombinant proteins; the proteins are only synthesized when the seeds are put in contact with water. Thus, recombinant human milk proteins can be introduced into the diet as rice in itself and be combined with various other food components (e.g. rice cereal), or a protein extract can be produced, yielding a product with higher protein and lower starch content.
We are using rice as an expression system to evaluate the biological activity of select human milk proteins. To date, we have expressed lactoferrin, lysozyme and ^-antitrypsin at very high levels, and large-scale field trials for several generations show that the transgenic rice is stable, expression levels are similar through generations, and the proteins are only expressed in the seeds. The genes were synthesized using codon optimization , i.e. the GC (guanine-cytosine) content was increased by nucleotide substitution but without changing any amino acid residue. Sequencing of the recombinant proteins confirmed that the amino acid sequence was identical to that of the native proteins. The signal sequence coding for storage was used. The gene was inserted by the so-called 'gene gun' technique and calli were grown in culture. Positive plants, detected by extraction and Western blots, were grown in greenhouses to obtain mature seeds. Seeds positive for the recombinant protein were subsequently grown in fields according to USDA regulations. Purified recombinant human lactoferrin and ^-antitrypsin were found to be glycosylated, but the carbohydrate content was less than that of their native counterparts. In other words, rice does recognize the signals for N-linked glycosylation and the specific sites were glycosylated, but the glycans are smaller than those in the native proteins. The carbohydrates in the rice glycans are similar to those in the human N-linked glycoproteins, i.e. man-nose and fucose. The terminal residues, however, are usually mannose or xylose, while the native proteins usually have fucose or sialic acid. This difference in terminal monosaccharides in the recombinant proteins may affect stability (e.g. liver clearance) when used for intravenous applications, but is unlikely to have an effect on food applications or to affect allergenicity. In the case of lactoferrin, which has its own specific receptor in the small intestine , we have shown that de-glycosylated human lactoferrin as well as different recombinant forms of human lactoferrin (Aspergillus, rice) bind equally well to the receptor, i.e. the glycan moiety of lactoferrin is not involved in the binding to the receptor. Thus, we conclude that the glycosylation of these human milk proteins when synthesized in rice is unlikely to affect their nutritional value.
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