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Department of Nutrition, University of California, Davis, California
Address correspondence to: Bo Lönnerdal, PhD, Department of Nutrition, University of California, Davis, CA 95616. E-mail: bllonnerdal{at}ucdavis.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONREFERENCES |
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1-antitrypsin in rice at very high expression levels. These recombinant proteins showed a stability and activities similar to those of the native milk proteins, suggesting that they may be able to exert biological activities in infants when added to formula or baby foods.
Key words: human milk proteins, recombinant milk proteins, plant expression
Key teaching points:
• Recombinant human milk proteins can be expressed in plants at high levels.
• Recombinant human milk proteins appear to have biological activities similar to those of the native proteins.
• Recombinant human milk proteins appear to have stability against heat, low pH and proteolytic degradation that is similar to that of the native proteins.
• Extensive safety and efficacy trials will be needed prior to the potential addition of these proteins to infant formula or baby foods.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONREFERENCES |
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Recombinant human milk proteins can now be expressed in a variety of systems [5]. Some of the first commercial applications were 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 breast milk, is being expressed in transgenic cows [6] and is now commercially available. Purified recombinant human lactoferrin is, however, too expensive to be used as a food additive or in infant formula. Cow’s milk containing recombinant human lactoferrin has not yet found a market, possibly due to logistics (transport, storage) and/or cost.
Microorganisms such as Saccharomyces and Aspergillus are also 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 [7]. Aspergillus, however, is successfully used for the production of recombinant human lactoferrin [8], 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. Pharmaceutical applications, being more cost tolerant, are actively pursued for this protein.
Expression of Human Milk Proteins in Plants
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.
Tobacco has been used for the expression of human lactoferrin [9]. Expression levels were modest, 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. To date, there have been no tests of activity of this protein, and it is not commercially available. Human lactoferrin has been expressed in potatoes [10], 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 [11,12] and is now being used for the expression of several human milk proteins, such as lactoferrin, lysozyme and 1-antitrypsin [13–18]. Very high expression levels can be achieved; for example 5 g of human lactoferrin per kg dehusked rice has been expressed in large scale field trials for several generations [14]. In fact, the higher level of expression turned the rice grains pink, due to the iron bound to lactoferrin. 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.
Expression of Human Milk Proteins in Rice
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 1-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 expressed only in the seeds. The genes were synthesized using codon-optimization [19]; that is, 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 alpha-1-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., mannose 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; that is, the glycan moiety of Lf 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.
Activity of Recombinant Human Milk Proteins Expressed in Rice
We have verified that the recombinant human milk proteins have their intended biological activities in vitro. Recombinant human lactoferrin was shown to both bind and release iron at low pH in a manner similar to that of native human lactoferrin [13]. It also bound similarly to the human lactoferrin receptor, which is present on the surface of the human intestinal cell line, Caco-2, grown in culture, and with an affinity similar to that of native lactoferrin. In addition, recombinant human lactoferrin was shown to inhibit the growth of enteropathogenic E. coli (EPEC), one of the most common causes of diarrhea in infants and children, and at a concentration similar to that of native human lactoferrin. When tested in the conventional assay for lysozyme activity, which is based on the lysis of Micrococcus lysodeiktus, the recombinant human lysozyme had an activity similar to that of native human lysozyme. The recombinant human lysozyme also inhibited the growth of EPEC at the same concentration as the native enzyme. Breast milk contains a significant concentration of 1-antitrypsin [20], and we have hypothesized that this inhibitor of proteolytic activity in the small intestine contributes to the "survival" of some human milk proteins, so that they can exert their physiological activities in the upper small intestine. The recombinant human 1-antitrypsin inhibited trypsin and elastase to an extent similar to that of native human 1-antitrypsin [18]. Thus, for these three human milk proteins we have demonstrated that they have activities similar to those of their native counterparts. For two of these proteins, lactoferrin and 1-antitrypsin, this occurred in spite of some differences in glycosylation.
Stability of Recombinant Human Milk Proteins
To be active in the small intestine, the recombinant proteins need to be able to withstand exposure to low pH in the stomach. While the gastric pH of infants rarely is below pH 4–5 for the first six months of life, in some cases the pH may be as low as pH 2–3. We therefore exposed the recombinant human milk proteins to low pH, from pH 2 to 5, for 30 minutes and then adjusted the pH back to neutral. Activities of all three recombinant proteins, assessed as above, were similar to those of the native proteins [13,15,18]. If recombinant human milk proteins are to be added to infant formula or baby foods, some degree of processing may be involved. We therefore exposed the recombinant proteins, both in pure form in solution and as added to infant formula, to various heat treatments, ranging from 78–100°C for 8 seconds up to 30 minutes. Except for the most severe treatment, 100°C for 5 minutes, which partially inactivated both recombinant and native human milk proteins, these proteins maintained activities similar to those of the native proteins [13,15,18].
Digestive Fate of Recombinant HumanMilk Proteins
Proteins are in general effectively digested by the combined proteolytic activities in the stomach and small intestine. Infants, however, are an exception from this. Infants have low pepsin secretion, and the pH of the infant stomach is usually around pH 4–5 [21], a pH too high for significant pepsin activity. In addition, secretion of pancreatic enzymes is immature, limiting the proteolytic activity in the small intestine. Further, some human milk proteins have structures that make them relatively resistant against proteolytic enzymes. As an illustration of this, we have found significant concentrations of lactoferrin, secretory IgA and 1-antitrypsin in the stool of breast-fed infants [20,22], and this persists up to at least four to six months of age. The proportion of proteins surviving decreases with increasing age, most likely as digestive function matures. It is, of course, also possible that some milk proteins survive passage of the stomach and the duodenum and may exert actions in the upper gut, to become digested further down in the small intestine.
We have developed an in vitro digestive system [23] to evaluate the survival of recombinant human milk proteins. Briefly, the proteins, in pure form or added to infant formula, are exposed to low pH and pepsin for 30 minutes at 37°C. The pH is then adjusted to pH 7 and pancreatin (mixture of pancreatic enzymes) is added. The solution is then incubated for 30 or 60 minutes, and the proteolytic enzymes are inactivated by brief (2–3 minutes) boiling. The molecular weights of the proteins are assessed by Western blotting, and their activities are assessed as described earlier. For all three proteins we studied, activities remained after treatment, and to an extent similar to that of the native human milk proteins. While some degradation did occur, the major part of the activity remained, and the extent of degradation was similar for recombinant and native proteins.
Further Testing
We have shown that recombinant human milk proteins can be produced at very high levels in rice and that they can withstand low pH and heat treatment, as well as proteolytic digestion in vitro. Further efficacy and safety trials in animals and humans will be needed next. Safety studies in rats will be necessary prior to human trials. For efficacy studies, we have chosen to first use a rat pup model, in which we can assess resistance against proteolytic activity as well as anti-infective properties (incubate with pathogens). We subsequently will use infant rhesus monkeys. This non-human primate model of human infants has a high degree of validity as their gastrointestinal physiology is very similar to that of human infants, and rhesus milk is reasonably similar to human milk both in nutrient content and with regard to bioactive proteins (e.g. lactoferrin). Another advantage with this model is that they can be fed regular infant formulas, without any modification of nutrient content, exclusively for four to six months. The addition of recombinant human milk proteins to infant formula can therefore be evaluated under realistic conditions. This model also allows us to infect the infant with a pathogen, such as EPEC, and evaluate the effect of the added protein on diarrhea prevalence, severity and duration [24]. Such studies, of course, are not possible to do in human infants. Once efficacy has been shown in these models, human trials are needed both with regard to efficacy and safety. For applications to infant formula the efficacy trials are essential as infant formulas are regulated by Federal law, the Infant Formula Act, which does not allow additions of novel components unless benefits have been demonstrated. Thus, addition of components primarily for marketing reasons, which occurs in some countries, will not be allowed.
Conclusions
Expression of recombinant human milk proteins in rice is realistic and a possibility for the addition of bioactive factors to infant formula and baby foods. We have been able to produce several such proteins at very high expression levels and have shown that the transmission is stable through several generations. Large-scale field trials have produced such rice in several tons, making it possible for evaluation of efficacy. In vitro studies have shown that these proteins have activities and stabilities similar to those of the native proteins and that they, to a considerable extent, withstand heat treatment. Further animal work and studies in human subjects are needed to document their efficacy and safety.
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