Abstract
The sustainable production of functional human proteins in plants offers a transformative path for developing animal-free nutritional and therapeutic compounds. In this study, we report one of the first expression of human α-lactalbumin (hLA), a key milk protein with nutritional and anticancer properties, via chloroplast genome engineering in Nicotiana tabacum. A codon-optimized hLA gene was introduced into the plastid genome using a synthetic expression cassette under the control of strong plastid regulatory elements. Homoplasmic transplastomic lines were obtained and confirmed via PCR and Southern blot analysis. Immunoblotting and ELISA quantification revealed that hLA accumulated to 23.4% of total soluble protein (TSP), one of the highest levels reported for plastid-expressed human proteins. Far-UV circular dichroism (CD) spectroscopy confirmed that the chloroplast-derived hLA adopted a native-like α-helical structure. Functionally, the recombinant protein successfully activated galactosyltransferase (GalT) in vitro, enabling lactose synthesis at 93% of the rate observed with native hLA. Furthermore, chloroplast-derived hLA was converted into a bioactive HAMLET complex by combining with oleic acid under mild thermal conditions. This complex induced potent apoptosis in human colorectal (WiDr) and breast cancer (MCF-7) cells, reducing viability to less than 8%, as confirmed by MTT and Annexin V/PI assays. These findings establish chloroplasts as an effective platform for high-yield, correctly folded, and functional production of human milk proteins. The ability to generate both enzymatically active and therapeutically functional products from a single plant-based system underscores its potential in food engineering and synthetic biology. This work offers a scalable, sustainable, and dual-purpose strategy for the development of recombinant milk proteins applicable in infant nutrition, functional foods, and plant-made biotherapeutics.
Data availability
The codon-optimized nucleotide sequence of the synthetic human α-lactalbumin (hLA) gene generated in this study is available in GenBank at NCBI under the accession number PX828338. All other datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.
References
Smith, N. W., Fletcher, A. J., Hill, J. P. & McNabb, W. C. Modeling the contribution of milk to global nutrition. Front. Nutr. 8, 716100 (2022).
Behm, K. et al. Comparison of carbon footprint and water scarcity footprint of milk protein produced by cellular agriculture and the dairy industry. Int. J. Life Cycle Assess. 27 (8), 1017–1034 (2022).
Andreas, N. J., Kampmann, B. & Le-Doare, K. M. Human breast milk: A review on its composition and bioactivity. Early Hum. Dev. 91 (11), 629–635 (2015).
Heine, W. E., Klein, P. D. & Reeds, P. J. The importance of α-lactalbumin in infant nutrition. J. Nutr. 121 (3), 277–283 (1991).
Permyakov, E. A. α-Lactalbumin, amazing calcium-binding protein. Biomolecules 10 (9), 1210 (2020).
Giuffrida, M. G. et al. The unusual amino acid triplet Asn-Ile-Cys is a glycosylation consensus site in human alpha-lactalbumin. J. Protein Chem. 16 (8), 747–753. https://doi.org/10.1023/a:1026359715821 (1997).
Kunz, C. & Lönnerdal, B. Re-evaluation of the Whey protein/casein ratio of human milk. Acta Paediatr. 81 (2), 107–112 (1992).
Layman, D. K., Lönnerdal, B. & Fernstrom, J. D. Applications for α-lactalbumin in human nutrition. Nutr. Rev. 76 (6), 444–460 (2018).
Enomoto, H. et al. Glycation and phosphorylation of α-lactalbumin by dry heating: effect on protein structure and physiological functions. J. Dairy Sci. 92 (7), 3057–3068 (2009).
Lien, E. L. Infant formulas with increased concentrations of α-lactalbumin. Am. J. Clin. Nutr. 77 (6), 1555S–1558S (2003).
Lönnerdal, B. Infant formula and infant nutrition: bioactive proteins of human milk and implications for composition of infant formulas. Am. J. Clin. Nutr. 99 (3), 712S–717S (2014).
Mossberg, A. K., Mok, H., Morozova-Roche, K., Svanborg, C. & L. A., & Structure and function of human α‐lactalbumin made lethal to tumor cells (HAMLET)‐type complexes. FEBS J. 277 (22), 4614–4625 (2010).
Permyakov, E. A. & Berliner, L. J. α-Lactalbumin: structure and function. FEBS Lett. 473 (3), 269–274 (2000).
Hettinga, K. & Bijl, E. Can Recombinant milk proteins replace those produced by animals? Curr. Opin. Biotechnol. 75, 102690 (2022).
Awasthi, V. et al. Contaminants in milk and impact of heating: an assessment study. Indian J. Public Health. 56 (1), 95–99 (2012).
Geistlinger, T. et al. Recombinant components and compositions for use in food products. Google Patents (2022).
Vestergaard, M., Chan, S. H. J. & Jensen, P. R. Can microbes compete with cows for sustainable protein production-A feasibility study on high quality protein. Sci. Rep. 6 (1), 36421 (2016).
Chaudhuri, T. K. et al. Effect of the extra N-terminal methionine residue on the stability and folding of Recombinant α-lactalbumin expressed in Escherichia coli. J. Mol. Biol. 285 (3), 1179–1194 (1999).
Overton, T. W. Recombinant protein production in bacterial hosts. Drug Discovery Today. 19 (5), 590–601 (2014).
Deng, M. et al. Efficient bioproduction of human milk Alpha-Lactalbumin in Komagataella phaffii. J. Agric. Food Chem. 70 (8), 2664–2672 (2022).
Saito, A., Usui, M., Song, Y., Azakami, H. & Kato, A. Secretion of glycosylated α-lactalbumin in yeast Pichia pastoris. J. Biochem. 132 (1), 77–82 (2002).
Demain, A. L. & Vaishnav, P. Production of Recombinant proteins by microbes and higher organisms. Biotechnol. Adv. 27 (3), 297–306 (2009).
Long, C. Transgenic livestock for agriculture and biomedical applications. In BMC Proceedings 8 (4), O29. https://doi.org/10.1186/1753-6561-8-S4-O29 (BioMed Central, 2014).
Bicar, E. H. et al. Transgenic maize endosperm containing a milk protein has improved amino acid balance. Transgenic Res. 17, 59–71 (2008).
Salmon, V. et al. Production of human lactoferrin in Transgenic tobacco plants. Protein Exp. Purif. 13 (1), 127–135 (1998).
Bock, R. Structure, function, and inheritance of plastid genomes. In Cell and Molecular Biology of Plastids 29–63. (Springer, 2007).
Chebolu, S. & Daniell, H. Chloroplast-derived vaccine antigens and biopharmaceuticals: expression, folding, assembly and functionality. Plant Produced Microb. Vaccines 33–54. (2009).
Koop, H. U., Herz, S., Golds, T. J. & Nickelsen, J. The genetic transformation of plastids. Cell Mol. Biol. Plastids 457–510. (2007).
Maliga, P. Engineering the plastid genome of higher plants. Curr. Opin. Plant. Biol. 5 (2), 164–172 (2002).
Oey, M., Lohse, M., Kreikemeyer, B. & Bock, R. Exhaustion of the Chloroplast protein synthesis capacity by massive expression of a highly stable protein antibiotic. Plant J. 57 (3), 436–445 (2009).
Oey, M., Lohse, M., Scharff, L. B., Kreikemeyer, B. & Bock, R. Plastid production of protein antibiotics against pneumonia via a new strategy for high-level expression of antimicrobial proteins. Proc. Natl. Acad. Sci. 106 (16), 6579–6584. (2009).
Daniell, H., Streatfield, S. J. & Wycoff, K. Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends Plant Sci. 6 (5), 219–226 (2001).
Fischer, R., Stoger, E., Schillberg, S., Christou, P. & Twyman, R. M. Plant-based production of biopharmaceuticals. Curr. Opin. Plant. Biol. 7 (2), 152–158 (2004).
Ma, J. K. C. et al. Molecular farming for new drugs and vaccines: current perspectives on the production of pharmaceuticals in Transgenic plants. EMBO Rep. 6 (7), 593–599 (2005).
Ehsasatvatan, M., Kohnehrouz, B. B., Gholizadeh, A., Ofoghi, H. & Shanehbandi, D. The production of the first functional antibody mimetic in higher plants: the Chloroplast makes the DARPin G3 for HER2 imaging in oncology. Biol. Res. 55 (1), 1–18 (2022b).
Zoubenko, O. V., Allison, L. A., Svab, Z. & Maliga, P. Efficient targeting of foreign genes into the tobacco plastid genome. Nucleic Acids Res. 22 (19), 3819–3824 (1994).
Ehsasatvatan, M., Kohnehrouz, B. B., Gholizadeh, A., Ofoghi, H. & Shanehbandi, D. Physical and biologically effective parameters in developing transplastomic tobacco plants by particle bombardment method using PDS-1000/He. Optimization 10 (2). (2022).
Murray, M. & Thompson, W. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 8 (19), 4321–4326 (1980).
Kamijima, T. et al. Heat-treatment method for producing fatty acid-bound alpha-lactalbumin that induces tumor cell death. Biochem. Biophys. Res. Commun. 376 (1), 211–214 (2008).
Žilinskas, J. et al. HAMLET effect on cell death and mitochondrial respiration in colorectal cancer cell lines with KRAS/BRAF mutations. J. Cancer Res. Clin. Oncol. 149 (11), 8619–8630 (2023).
Miller, E. Apoptosis measurement by Annexin V staining. Cancer Cell. Culture Methods Protoc. 191–202. (2004).
Jensen, R. G. Activation of Rubisco regulates photosynthesis at high temperature and CO2. Proc. Natl. Acad. Sci. 97 (24), 12937–12938. (2000).
Krishnan, H. B. & Natarajan, S. S. A rapid method for depletion of Rubisco from soybean (Glycine max) leaf for proteomic analysis of lower abundance proteins. Phytochemistry 70 (17–18), 1958–1964 (2009).
Ramboarina, S. & Redfield, C. Structural characterisation of the human alpha-lactalbumin molten globule at high temperature. J. Mol. Biol. 330 (5), 1177–1188. https://doi.org/10.1016/s0022-2836(03)00639-9 (2003).
Piazenski, I. N. et al. From lab to table: the path of recombinant milk proteins in transforming dairy production. Trends Food Sci. Technol. 104562. (2024).
Batt, C. A., Rabson, L. D., Wong, D. W. & Kinsella, J. E. Expression of Recombinant bovine β-lactoglobulin in Escherichia coli. Agric. Biol. Chem. 54 (4), 949–955 (1990).
Goda, S. et al. Recombinant expression analysis of natural and synthetic bovine alpha-casein in Escherichia coli. Appl. Microbiol. Biotechnol. 54, 671–676 (2000).
Kim, Y. et al. High-level expression of human α s1-casein in Escherichia coli. Biotechnol. Tech. 11, 675–678 (1997).
Kalidas, C., Joshi, L. & Batt, C. Characterization of glycosylated variants of β-lactoglobulin expressed in Pichia pastoris. Protein Eng. 14 (3), 201–207 (2001).
Kim, Y. K., Yu, D. Y., Kang, H. A., Yoon, S. & Chung, B. H. Secretory expression of human $\alpha_ {s1} $-Casein in Saccharomyces cerevisiae. J. Microbiol. Biotechnol. 9 (2), 196–200 (1999).
Totsuka, M. et al. Expression and secretion of bovine β-lactoglobulin in Saccharomyces cerevisiae. Agric. Biol. Chem. 54 (12), 3111–3116 (1990).
Sun, X. L., Baker, H. M., Shewry, S. C., Jameson, G. B. & Baker, E. N. Structure of Recombinant human lactoferrin expressed in Aspergillus Awamori. Acta Crystallogr., Sect D: Biol. Crystallogr. 55 (2), 403–407 (1999).
Chong, D. et al. Expression of the human milk protein β-casein in Transgenic potato plants. Transgenic Res. 6, 289–296 (1997).
Huang, N., Rodriguez, R. L. & Hagie, F. E. Expression of human milk proteins in transgenic plants. Google Patents (2014).
Lanquar, V. & Magi, E. R. Recombinant milk proteins and food compositions comprising the same. Google Patents (2022).
Tobin, C. J. Recombinant micelle and method of in vivo assembly. Google Patents (2022).
Ehsasatvatan, M. & Kohnehrouz, B. B. The lyophilized chloroplasts store synthetic DARPin G3 as bioactive encapsulated organelles. J. Biol. Eng. 17 (1), 63 (2023).
Scotti, N., Bellucci, M. & Cardi, T. The chloroplasts as platform for Recombinant proteins production. In Translation in Mitochondria and Other Organelles 225–262. (Springer, 2013).
Boyhan, D. & Daniell, H. Low-cost production of proinsulin in tobacco and lettuce chloroplasts for injectable or oral delivery of functional insulin and C‐peptide. Plant Biotechnol. J. 9 (5), 585–598 (2011).
Hakansson, A., Zhivotovsky, B., Orrenius, S., Sabharwal, H. & Svanborg, C. Apoptosis induced by a human milk protein. Proc. Natl. Acad. Sci. 92 (17), 8064–8068 (1995).
Ho Cs, J., Rydstrom, A., Manimekalai, M. S. S., Svanborg, C. & Grüber, G. Low resolution solution structure of HAMLET and the importance of its alpha-domains in tumoricidal activity. PloS ONE 7 (12), e53051. (2012).
Svensson, M. et al. Molecular characterization of α–lactalbumin folding variants that induce apoptosis in tumor cells. J. Biol. Chem. 274 (10), 6388–6396 (1999).
Funding
This work is based upon research funded by Iran National Science Foundation (INSF) under project NO. 4027239.
Author information
Authors and Affiliations
Contributions
M.E., designed the study and experiments, performed the experiments, analyzed the data, and wrote the manuscript. B. B. K., conceived, designed, organized, and supervised the study, provided specialized scientific and technical support, and revised the paper. All authors read and approved the final manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
About this article
Cite this article
Ehsasatvatan, M., Kohnehrouz, B.B. Transplastomic biofactory for the production of functional human α-lactalbumin for nutritional and therapeutic applications. Sci Rep (2026). https://doi.org/10.1038/s41598-026-38965-y
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41598-026-38965-y
