Lactoferrin (LF), a multifunctional glycoprotein of the transferrin family, is naturally expressed in human and cow milk. The name ‘LF’ is derived from its ability to bind to iron (ferrin, a suffix indicating iron-binding protein).
LF is a vital bioactive component in human milk and helps promote infant health and development. Scientists have been fascinated with LF since its discovery in the 1930s due to its unique biological features. LF plays a role in the immune response, antibacterial activity, and anti-inflammatory effects, among other biological activities.
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LF contributes to the innate immune response and acts as the first line of defense. It efficiently disrupts the integrity of cell membranes, restricting bacterial multiplication by reducing iron availability. LF also exerts antiviral activity against a variety of viruses. In addition, LF interacts with immune cells such as macrophages and lymphocytes and boosts the immune response. Hence, LF offers a diverse range of applications across the food, cosmetic, and pharmaceutical sectors.
Market demand
However, there are certain limitations in separating and purifying LF from milk, resulting in an inability to meet the current market demand with this product. To overcome these challenges, researchers have developed new technologies where microorganisms can synthesize LF via genetic engineering. Thanks to the development of such synthetic biological systems, it is now more feasible to use microorganisms to manufacture large volumes of LF.
In a recent study published on 20 August 2024, in Volume 6 of BioDesign Research, a team of scientists led by Dr. Kun Liu from Anhui Polytechnic University, China, discussed the design and construction of LF expression systems, including the associated challenges, solutions, and opportunities.
“The challenge in obtaining LF for market needs can be overcome by producing LF on an industrial scale through the use of synthetic biological expression systems as the structure of LF expressed in these systems is very similar to that of natural LF,” says Liu.
Structural properties
In order to create the synthetic systems of LF, it is important to know its structural properties. LF has a molecular weight of approximately 80 kilodalton and consists of roughly 700 amino acids. It has the C- and N-terminal lobes. The N-terminal lobe, which is less stable thermodynamically, houses the iron-binding site. Increasing or adding extra disulfide bridges to the N-terminal lobe can enhance LF’s thermostability.
LF is a protein with carbohydrates attached to its nitrogen atom through a process called N-linked glycosylation. The LF glycosylation increases resistance to enzyme degradation and thus maintains its structural stability. Therefore, achieving high stability in LF depends on using a suitable host-expression system.
In this study, researchers have summarized the four synthetic host biological systems—bacteria, yeast, filamentous mold, and cell lines—for producing high-expression LF. They also discussed the challenges and solutions for constructing high-yielding LF in these systems.
Biological system choices
In bacterial host systems, E. coli is the most popular synthetic biological system used for LF production. The E. coli system can produce 700 mg/L of human LF. However, there are certain limitations to it. The protein degrading activity of E. coli can harm the LF proteins, and the bacterial host lacks the machinery for biochemical modifications. Besides this, the newly synthesized LF proteins can cause harm to the host.
Compared to the bacterial system, yeast and mold are more competitive choices. Both the host systems offer strong LF expression and can perform biochemical processing, making a more stable LF protein. However, the main obstacle with these systems is that the newly produced LF can cause toxicity to the systems, thus limiting its expression.
The researchers emphasized reducing the LF toxicity during fermentation in order to increase protein expression. The final host system, cell lines, can potentially synthesize LF that is very consistent in both structure and function with natural LF. Adding further, Zhen Tong, the first co-first author, explains, “The main challenges with cell line systems are their high cultural costs, susceptibility to contamination, and ability to carry human pathogens. Moreover, the use of cell lines in large-scale LF production is still limited.”
Overcoming challengessyn
Talking about a way in which the challenges associated with synthetic biological systems can be overcome, Xuanqi Zhang, also affiliated with Anhui Polytechnique University, says, “It is important to redesign the transport mechanism of the expression host to ensure quick secretion of the produced LF into the extracellular environment. We should also consider knocking out key enzymes that can degrade LF in the host.”
In conclusion, the use of synthetic biological systems can help resolve the issue of obtaining LF on an industrial scale. By enabling the controlled production of LF through genetic engineering and host-organism interactions, these systems can open doors to applications in food, pharmaceuticals, and other sectors.
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