Expression and characterization of recombinant Müllerian Inhibiting Substance (MIS) using the Bombyx Mori Baculovirus – insect cell expression systemAbstract
The Müllerian inhibiting substance (MIS) is a glycoprotein that belongs to the transforming growth factor-β family and is known for its crucial roles in embryonic development and cellular differentiation. Attempts to produce recombinant MIS using bacterial and yeast expression systems have faced challenges, including insufficient post-translational modifications and low expression efficiency. In this study, the MIS gene was successfully cloned into the pBacPAK8 transfer vector and expressed using the Bombyx mori baculovirus - insect cell expression system. Co-transfection of BMN1 insect cells with the recombinant plasmid and baculoviral DNA led to the successful generation of recombinant baculoviruses. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis and Western blot analyses of lysates from infected cells confirmed the expression of a ~70 kDa MIS protein with significant antigenicity. To the best of our knowledge, this is the first report describing the recombinant production of human MIS protein using the B. mori baculovirus system. This expression platform provides a promising approach for synthesizing structurally complex human proteins and lays the groundwork for future research and biomedical applications.
Keywords:
Müllerian inhibiting substance; Bombyx mori; Baculovirus; recombinant protein; BMN1 cells
Resumo
A substância inibidora Mülleriana (MIS) é uma glicoproteína pertencente à família do fator de crescimento transformador-β (TGF-β), crucial para o desenvolvimento embrionário e diferenciação celular. Tentativas anteriores de produzir MIS recombinante em sistemas de expressão bacterianos e de levedura enfrentaram limitações, como modificações pós-traducionais inadequadas e baixos rendimentos. Neste estudo, o gene MIS humano foi clonado com sucesso no vetor de transferência pBacPAK8 e expresso usando o sistema de expressão baculovírus–célula de inseto Bombyx mori. A cotransfecção de células de inseto BMN1 com o plasmídeo recombinante e o DNA baculoviral resultou na geração bem-sucedida de baculovírus recombinantes. As análises por eletroforese em gel de poliacrilamida com dodecilsulfato de sódio (SDS-PAGE) e Western blot dos lisados de células infectadas confirmaram a expressão de uma proteína MIS de ~70 kDa com antigenicidade significativa. De acordo com o nosso conhecimento, este é o primeiro relato de produção de MIS humana utilizando o sistema de baculovírus B. mori. Esta plataforma oferece uma abordagem promissora para a síntese de proteínas humanas complexas, fornecendo uma base para futuras aplicações biomédicas.
Palavras-chave:
Substância inibidora Mülleriana; Bombyx mori; Baculovírus; proteína recombinante; Células BMN1
1. Introduction
Müllerian inhibiting substance (MIS) is a glycoprotein with a molecular mass of approximately 70 kDa, containing about 15% carbohydrate and consisting of two disulfide-linked monomers. Each monomer includes an amino-terminal domain (~57 kDa) and a carboxyl-terminal domain (~12.5 kDa). As a member of the transforming growth factor-β superfamily, MIS is classified within the cytokine family and exhibits growth-regulatory properties (Kim et al., 2014; MacLaughlin and Donahoe, 2010; Kim et al., 2021; Pieretti-Vanmarcke et al., 2006; Bedenk et al., 2020; Rak et al., 2016).
Genetic studies have shown that the human MIS gene is located on chromosome 19, region p13.3–p13.2 (Lindhardt Johansen et al., 2013; Nathalie Josso et al., 2001). Analysis of the protein encoded by this gene has revealed that it consists of a polypeptide chain of 560 amino acids (Kim et al., 2014).
MIS plays a key role during mammalian embryonic development by regulating the regression of the Müllerian ducts in male embryos, thereby preventing the development of female reproductive organs. In addition, MIS participates in several physiological processes related to cell differentiation, growth, and apoptosis (Zhao and Yao, 2019; Rey and Grinspon, 2024; Mullen and Behringer, 2014).
In recent years, MIS has gained considerable interest as a potential agent for clinical and biotechnological applications, particularly in reproductive medicine and oncology (MacLaughlin and Donahoe, 2010; Gowkielewicz et al., 2024; Pépin et al., 2018; Kushnir et al., 2017; Li and Nelson, 2020).
However, obtaining biologically active recombinant MIS protein has been challenging. Attempts to express MIS in bacterial and yeast expression systems have been largely unsuccessful due to improper protein folding and the absence of essential post-translational modifications. Mammalian expression systems, including Chinese hamster ovary (CHO) cells, have been employed to produce recombinant human MIS. Although these systems enable proper post-translational modifications, they are limited by low expression yields, high production costs, and complex cultivation requirements (Kim et al., 2021; Chin et al., 1991; Li et al., 2022; Geng et al., 2024).
Among the various expression systems used for recombinant protein production, the baculovirus–insect cell system (baculovirus expression vector system, BEVS) is considered one of the most efficient platforms (Felberbaum, 2015; Hitchman et al., 2009; Sułek and Szuster-Ciesielska, 2025). This is largely because of its relatively low production costs combined with high protein yields (Kato et al., 2005). In addition, the system supports proper post-translational processing and modification, making it particularly suitable for producing complex, biologically active proteins (Yamaji, 2011; Sookhoo et al., 2024; Pidre et al., 2022).
Most applications of this system for recombinant protein expression have used recombinant baculoviruses based on the Bombyx mori nucleopolyhedrovirus (BmNPV). These viruses are highly specific, infecting only a limited range of insects and posing no risk to humans or livestock. Although recombinant protein expression can be performed in B. mori insect cell cultures in vitro, this approach remains relatively expensive because it requires enriched media and tightly controlled growth conditions. In contrast, the direct use of silkworm larvae as whole-organism “bioreactors” provides a cost-effective alternative, eliminating the need for specialized culture media and equipment and significantly reducing the overall production cost of recombinant proteins (Motohashi et al., 2005; Xu et al., 2019; Mao et al., 2018).
Therefore, this study aimed, for the first time, to construct a recombinant BmNPV carrying the human MIS gene and to express and characterize the recombinant MIS protein in B. mori BMN1 insect cells.
2. Materials and Methods
2.1. The following laboratory equipment was used for all experimental procedures
Class II biosafety cabinet (Thermo Electron Corporation, Germany; Model: КС 15 1/ПЕ AC), CO2 incubator (ТСО-1/80 СПУ, Russia), Refrigerators and freezers (+4 °C to -20 °C), Electronic pH meter (METTLER TOLEDO SevenEasy), Electronic balance (Sartorius), Spectrophotometer (Analytik Jena, SPECOL 1300), Vortex mixer (Stuart Scientific, VibroGENE CA4), Refrigerated centrifuge (Jouan MR 1812), Mini-centrifuge (Microspin 12, Biosan), Real-time polymerase chain reaction (PCR) system (StepOne, Applied Biosystems), Electrophoresis system with power supply (Bio-Rad PAC 1000), Microscope (Leica DMIL), Electroporator (Eppendorf 2510), RNA/DNA calculator.
2.2. Plasticware and Glassware
Cell culture flasks (Thermo Scientific, 25 cm2 and 75 cm2; Cat. Nos. 690160, 658170), Glass pipettes (Thermo Scientific, 5 ml and 10 ml; Cat. No. 401746 7740), Polypropylene tubes (Thermo Scientific, 50 ml; Cat. No. 2098), Polystyrene tubes (Becton Dickinson, 12 × 75 mm, 5 ml; Cat. No. 2063), 24-well polystyrene plates (Thermo Scientific; Cat. No. 3524), 0.22 μm filters (Pall Corporation; Cat. No. 20624).
2.3. Media and reagents
Grace's insect cell culture medium (HiMedia, India; Cat. No. 11590-056), Fetal bovine serum (FBS, heat-inactivated), LB broth and LB agar for Escherichia coli cultures (Merck, Germany; Invitrogen, USA), PBS-EDTA buffer for insect cell lysis (pH 6.2), TAE buffer for electrophoresis, Loading buffer and ethidium bromide solution (10 mg/ml), Agarose for electrophoresis and plaque assays, Antibiotics: penicillin G and streptomycin, Transfection reagents: ExGen 500 (Fermentas, USA; Cat. No. P0511) and Metafectene® PRO (Biontech, Germany; Cat. No. T040).
2.4. Cell lines and plasmids
E. coli NEB-5α competent cells for molecular cloning, Recombinant plasmid containing the human MIS gene, B. mori (BMN1) insect cell line for recombinant protein expression.
2.4.1. Bacterial strains, plasmids, and cell lines
Competent E. coli NEB-5α cells (New England Biolabs, USA) were used for plasmid amplification and cloning. The donor plasmid pBs-Ac-polh-MIS, containing the human MIS gene under the control of the polyhedrin promoter, was used as the source of the MIS gene. The pBacPAK8 transfer vector (Clontech, USA) was used to generate recombinant baculovirus constructs (Sasmakov et al., 2021)
For recombinant protein expression, B. mori BMN1 insect cells were used. Cells were maintained in Grace’s insect cell culture medium (HiMedia, India) supplemented with 10% fetal bovine serum (Gibco, USA) at 26 °C.
2.5. Isolation and preparation of the MIS gene
Plasmid DNA containing the MIS gene (pBs-Ac-polh-MIS) was extracted from E. coli using the standard alkaline lysis method. The plasmid was digested with EcoRI restriction enzyme (Thermo Scientific, USA) according to its physical map to isolate the MIS gene fragment with cohesive ends. The resulting DNA fragment was purified by agarose gel electrophoresis followed by gel extraction.
2.6. Construction of recombinant plasmid
The purified MIS gene fragment was ligated into the EcoRI site of the pBacPAK8 transfer vector using T4 DNA ligase (Thermo Scientific, USA). The ligation mixture was introduced into electrocompetent E. coli NEB-5α cells by electroporation. Transformants were selected on LB agar plates containing ampicillin (100 µg/ml) (Abdurakhmanov et al., 2023; Fateev et al., 2024).
2.6.1. Screening and confirmation of recombinant plasmid
Positive colonies were screened by colony PCR using MIS gene–specific primers. PCR products were analyzed by agarose gel electrophoresis. The presence and orientation of the MIS insert were further confirmed by restriction enzyme digestion followed by gel electrophoresis.
2.7. Isolation of Baculovirus DNA
BmNPV baculovirus DNA was extracted from infected B. mori cell cultures by sucrose cushion centrifugation followed by phenol–chloroform extraction. DNA purity and concentration were assessed spectrophotometrically.
2.7.1. Cell culture and transfection of BMN1 cells
BMN1 insect cells were cultured in Grace’s medium supplemented with 10% FBS at 26°C. For recombinant virus generation, BMN1 cells were co-transfected with the recombinant plasmid pBacPAK8-MIS and BmNPV viral DNA using ExGen 500 (Fermentas, USA) or Metafectene® PRO (Biontech, Germany) transfection reagents. After three days of incubation, recombinant baculoviruses were harvested from the culture supernatant.
2.7.2. Plaque assay and virus titration
Recombinant baculoviruses were isolated by plaque assay using BMN1 cell monolayers. Virus titers were determined by the TCID50 method in 96-well plates.
2.7.3. Infection of BMN1 cells and recombinant protein expression
BMN1 cells were infected with the recombinant baculovirus at the appropriate multiplicity of infection. After 72 hours of incubation at 26 °C, cells were harvested for recombinant MIS protein expression analysis.
2.8. Protein extraction and SDS-PAGE
Infected and uninfected BMN1 cells were lysed using standard lysis buffer. Total protein extracts were prepared, and protein samples were separated on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels. Gels were stained with Coomassie Brilliant Blue to visualize protein bands.
2.8.1. Western blotting and immunodetection
For specific detection of the recombinant MIS protein, Western blotting was performed. After electrophoresis, proteins were transferred onto nitrocellulose membranes. Membranes were blocked with 5% non-fat dry milk in TBST and incubated overnight at 4 °C with the commercial monoclonal antibody “Monoclonal Antibody to Anti-Müllerian Hormone (AMH)” (Cloud-Clone Corp., Cat. No. MAA228Hu22). After washing, membranes were incubated with HRP-conjugated secondary antibodies (Santa Cruz Biotechnology, USA), and bands were visualized using a chromogenic substrate (Azimova et al., 2023).
2.9. Cell viability assessment
Cell viability during culture and infection was determined by trypan blue exclusion using a hemocytometer.
2.10. Protein modeling and in silico structural analysis
To confirm that the recombinant Müllerian Inhibiting Substance (MIS) expressed in Bombyx mori retained its native receptor-binding conformation, an in silico structural validation was performed. Because the biological activity of MIS depends critically on correct folding and its specific interaction with the AMH type II receptor (AMHR2), this computational approach was applied to verify that expression in insect cells did not alter its native three-dimensional configuration. The amino acid sequence of recombinant MIS was derived from the gene cloned and deposited in the NCBI GenBank (accession no. MZ556958.1), which represents a synthetic construct designed for heterologous expression. Sequence alignment confirmed that the translated amino acid sequence of MZ556958.1 is 100% identical to the human AMH reference sequence (Uniprot: P03971). Consequently, the high-confidence structural model (AF-U3GVP6-F1) from the AlphaFold Protein Structure Database was utilized as a structural template for the recombinant protein. The experimentally determined human AMH–AMHR2 complex (PDB ID: 7L0J) was retrieved from the Protein Data Bank for comparative modeling. Both structures were analyzed using PyMOL 3.1 (Schrödinger LLC) to visualize domain organization, identify potential ligand–receptor interfaces, and highlight conserved residues. Key amino acids at the interface—K534 (ligand) and D81/E84 (receptor)—were specifically examined to assess possible salt-bridge and hydrogen-bond interactions. This type of comparative molecular analysis has not been previously applied to MIS expressed in a non-mammalian expression system, making this study the first to evaluate the receptor-binding potential of insect-derived recombinant MIS.
3. Results
To express the MIS protein in B. mori (BMN1) cells, the pBacPAK8 transfer vector, derived from the BmNPV, was selected. The pBacPAK8 plasmid is 5538 base pairs (bp) in length and contains genomic elements derived from B. mori, including the strong polyhedrin (polh) gene promoter, which enables high-level expression of the target protein.
To insert the MIS gene into the pBacPAK8 transfer vector, restriction digestion was performed with EcoRI according to the physical map of the plasmid (Figure 1).
EcoRI recognition sequence: 5′–G↓AATTC–3′
3′–CTTAA↓G–5′
A – Linearized form of the pBacPAK8 transfer vector following digestion with EcoRI;
B – Circular (supercoiled) form of the pBacPAK8 plasmid.
Restriction digestion products were analyzed by 0.8% agarose gel electrophoresis, which confirmed that the linearized pBacPAK8 vector was approximately 5538 bp in length.
The donor plasmid pBs-Ac-polh-MIS, containing the full-length MIS gene under the control of the polyhedrin promoter, was digested with EcoRI. Electrophoresis revealed two DNA fragments: a 3651 bp vector backbone and a 2058 bp MIS gene insert, confirming the presence of the gene (Figure 2).
As shown in Figure 2, A – Linear form of the undigested pBs-Ac-polh-MIS plasmid. B and C – DNA fragments generated after EcoRI digestion: B – 3651 bp pBs-Ac-polh vector fragment; C – 2058 bp MIS gene fragment. The MIS gene fragment was subsequently excised and purified from a 0.8% agarose gel under homogeneous conditions.
To prevent self-ligation of the vector, the digested ends were treated with alkaline phosphatase. The resulting DNA fragments were ligated using T4 DNA ligase at a 1:3 molar ratio (vector:insert) (Figure 3).
Following ligation, the recombinant pBacPAK8-polh-MIS plasmid containing the MIS gene was successfully obtained. This plasmid was then introduced into E. coli NEB-5α cells by electroporation.
Because the recombinant plasmid carries an ampicillin resistance gene, only bacterial clones harboring the construct grew on LB agar plates supplemented with ampicillin (100 µg/ml), allowing for efficient selection of positive transformants.
To confirm the presence and integrity of the MIS gene in the recombinant pBacPAK8-polh-MIS plasmid, PCR analysis was performed using four pairs of gene-specific primers designed based on the nucleotide sequence of the human MIS gene retrieved from the NCBI database. The primers were synthesized using a Synthesizer DNA/RNA ASM-2000 device and purified by 20% polyacrylamide gel electrophoresis.
Each primer pair amplified a specific region of the gene, and PCR was performed under standard conditions. The resulting amplification products were analyzed on a 1% agarose gel (Figures 4-5). The details of the primers and their expected amplicon sizes are presented in Table 1.
Schematic representation of primer binding sites across the MIS gene sequence used for PCR screening.
Agarose gel electrophoresis of PCR products amplified from the recombinant pBacPAK8-polh-MIS plasmid using four internal primer sets.
The MIS gene in the recombinant pBacPAK8-polh-MIS plasmid is 1683 bp in length. PCR screening was performed using four primer sets targeting different regions of the gene (see Table 1). Agarose gel electrophoresis revealed amplification products of 538 bp, 433 bp, 459 bp, and 719 bp, each matching the expected amplicon size. These results confirm that all targeted regions of the MIS gene were successfully amplified, demonstrating that the entire coding sequence is present and correctly integrated within the recombinant plasmid.
To verify the orientation of the MIS gene within the vector, PCR was performed using a forward primer (Poly-F) specific to the polyhedrin promoter region and a reverse primer (MIS-R5) specific to the MIS gene. Amplification of a 513 bp product confirmed the correct directional insertion of the gene into the vector (Figures 6-7).
Schematic location of Poly-F and MIS-R5 primers confirming the orientation of the MIS gene in the vector.
PCR confirmation of the orientation of the MIS gene within the recombinant pBacPAK8-polh-MIS plasmid.
Poly-F: 5′-TTGTTAAAAATAACAGCCATT-3′
MIS-R5: 5′-TCTAAGCGCCTATGAGCA-3′
PCR analysis was performed using a forward primer specific to the polyhedrin promoter region and a reverse primer specific to the MIS gene. The expected amplicon size for a correctly oriented insert was 513 bp, whereas an incorrectly oriented insert would have yielded a 1441 bp product.
As shown in the gel image, a single 513 bp amplification product was observed, corresponding to the theoretical size expected for the correct orientation. No nonspecific bands or larger fragments were detected.
These results confirm that the MIS gene was successfully cloned in the correct orientation downstream of the polyhedrin promoter in the recombinant pBacPAK8-polh-MIS plasmid, ensuring proper transcription and expression in subsequent steps.
The recombinant pBacPAK8-polh-MIS plasmid and wild-type BmNPV viral DNA were co-transfected into B. mori BMN1 cells using Metafectene® PRO. Optimal co-transfection conditions were established as follows: 2.0 µg of plasmid DNA, 1.0 µg of viral DNA, and 10 µl of transfection reagent per 3 µg of total DNA. After seven days, recombinant rBmNPV-MIS virus stocks were isolated.
Purification of the recombinant baculovirus was performed by plaque assay. Virus clones underwent three rounds of purification and were subsequently titrated using the TCID50 method. In addition, viral genome copy number was determined by quantitative PCR (qPCR). The recombinant virus stock contained approximately 2 × 106 genome copies/ml (Figure 8).
Quantitative PCR (qPCR) analysis of the MIS gene region in recombinant baculovirus preparations.
Quantitative PCR (qPCR) was performed to determine the viral genome copy number in recombinant baculovirus stocks containing the MIS gene. A standard curve was generated using three reference solutions with known concentrations: (1) 2 × 103 copies/ml, (2) 2 × 104 copies/ml, and (3) 2 × 105 copies/ml.
Samples under investigation are represented as points 4 and 5 (green lines) on the graph. Based on comparison with the standard curve, the viral concentration in these samples was calculated to be approximately 2 × 106 copies/ml.
These results confirm that the isolated recombinant baculovirus clones contained sufficient viral particles, making them suitable for infection of insect cell lines in downstream protein expression experiments.
The recombinant baculovirus rBm-polh-MIS was used to infect B. mori BMN1 insect cell cultures. The infected cells were incubated at 22–26°C in Grace’s medium supplemented with 10% FBS. After five days of incubation, cells were harvested by centrifugation and analyzed for recombinant protein expression.
Cell lysates from infected and control BMN1 cultures were prepared and subjected to denaturing SDS-PAGE on a 10% polyacrylamide gel to assess recombinant protein expression. A distinct protein band was observed in the lysate of infected cells at a molecular weight of approximately 70 kDa (Figure 9), corresponding to the predicted size of the MIS monomer based on its amino acid sequence. No comparable band was detected in the uninfected control samples.
A: Lysate from cells infected with rBm-polh-MIS showing a strong ~70 kDa band corresponding to recombinant MIS. B: Lysate from uninfected BMN1 control cells.
To confirm the identity of the expressed protein, Western blotting was performed using a commercial monoclonal antibody specific for human MIS AMH (Cloud-Clone Corp., Cat. No. MAA228Hu22).
A single immunoreactive band was detected at approximately 70 kDa in the infected sample, confirming that the expressed protein was recombinant MIS and that it retained its antigenic specificity (Figure 10). No signal was observed in the control lysates.
Western blot analysis of recombinant MIS protein expressed in B. mori BMN1 cells using an anti-AMH monoclonal antibody.
These results demonstrate that the recombinant MIS protein was successfully expressed in BMN1 cells, migrated at approximately 70 kDa, and reacted specifically with the anti-human MIS monoclonal antibody. This confirms not only the correct expression but also the preservation of its antigenic properties, supporting its suitability for further functional and biomedical studies.
The predicted 3D structure of recombinant MIS showed the typical cystine-knot topology characteristic of the TGF-β superfamily, indicating that the protein was likely folded correctly. When aligned with the experimentally determined human AMH–AMHR2 complex (PDB ID: 7L0J), the recombinant model displayed a high degree of structural similarity, particularly in the receptor-binding region. Key residues at the ligand–receptor interface — K534 of MIS and D81/E84 of AMHR2—were identified as conserved amino acids involved in potential salt-bridge and hydrogen-bond formation. Visualization in PyMOL revealed spatial proximity between these residues, forming two specific contacts: K534–D81 (electrostatic) and K534–E84 (hydrogen bond). These findings indicate that the recombinant MIS expressed in Bombyx mori cells preserves the structural determinants required for AMHR2 recognition. These findings suggest that the recombinant MIS expressed in Bombyx mori cells retains key predicted structural features associated with AMHR2 recognition. Importantly, previous studies have analyzed these interactions only in mammalian-expressed AMH proteins, while our study provides the first evidence—through in silico modeling—that insect-derived recombinant MIS can maintain an equivalent receptor-binding configuration. This validation supports the structural reliability of the B. mori expression platform for producing complex human glycoproteins (Figure 11A, B).
Combined in silico structural model of recombinant MIS and its receptor interaction sites.
(A) Predicted 3D structure of recombinant Müllerian Inhibiting Substance (MIS) modeled using AlphaFold (AF-U3GVP6-F1-model_v4) and aligned with the experimentally determined AMH–AMHR2 complex (PDB ID: 7L0J). Chain A (ligand, MIS) is shown in light blue and Chain B (receptor, AMHR2) in soft pink. (B) Visualization of ligand–receptor interface showing conserved electrostatic and hydrogen-bond contacts between K534 (ligand) and D81/E84 (receptor), represented by orange and yellow lines, respectively. These in silico analyses confirm that the recombinant MIS expressed in Bombyx mori retains a native-like receptor-binding conformation consistent with the human AMH structure.
4. Discussion
The production of bioactive recombinant human MIS protein has historically posed a significant challenge due to its structural complexity and requirement for accurate eukaryotic post-translational modifications. Prokaryotic systems such as E. coli and yeast often fail to achieve proper folding and glycosylation of MIS, resulting in inactive or misfolded products (Liu et al., 2016; Yagi et al., 2020; Kato et al., 2010). Mammalian expression systems, including CHO and HEK293 cells, can produce correctly folded MIS but are cost-intensive, yield less than 1 mg/l, and require complex culture conditions (Portolano et al., 2014; Rosini and Pollegioni, 2023).
The baculovirus–insect cell system represents a promising alternative, as it supports high-level expression and eukaryotic protein processing (Wang et al., 2024). Most previous studies have employed Sf9 or Hi5 cell lines derived from Spodoptera frugiperda or Trichoplusia ni, respectively (Wilde et al., 2014; He et al., 2023). Although these systems are efficient, they require serum-free conditions and may present scalability limitations.
In contrast, our study demonstrates the feasibility of using the B. mori (silkworm) baculovirus system to express full-length human MIS protein. To our knowledge, this is the first report of MIS expression in B. mori BMN1 cells using the pBacPAK8 vector. A key advantage of this platform is its compatibility with in vivo larval-scale expression, which allows entire silkworms to be used as bioreactors. Previous studies with other recombinant proteins expressed in B. mori larvae have reported yields of up to 10–20 mg per larva, which represents a substantially higher output compared with cell culture systems (Yu et al., 2021; Kang et al., 2023).
Our results confirmed the production of a ~70 kDa MIS protein with strong immunoreactivity using monoclonal antibodies specific for human AMH, indicating that the recombinant protein retains its structural and antigenic integrity.
Although the present study did not assess the biological functionality of MIS (e.g., receptor interaction or in vitro bioassays), the established expression system provides a robust platform for future functional and therapeutic investigations.
To further support the structural integrity and receptor-binding potential of the recombinant MIS, in silico molecular modeling was performed. The tertiary structure predicted by AlphaFold was superimposed on the experimentally determined AMH–AMHR2 complex, revealing strong structural alignment. Key interfacial residues (K534 in MIS and D81/E84 in AMHR2) were preserved, indicating that the insect-derived recombinant MIS retained its essential receptor-binding domains. Previous studies (Hart et al., 2021) similar receptor interfaces in human AMH proteins expressed in mammalian systems. However, the present study is the first to demonstrate, through computational validation, that recombinant MIS produced in Bombyx mori insect cells can achieve the same structural fidelity as the native human hormone.
These findings support the feasibility of insect-based expression systems for producing structurally complex recombinant human proteins and provide a foundation for future functional studies.
5. Conclusion
In this study, we successfully achieved recombinant expression and characterization of human MIS protein using the B. mori baculovirus–insect cell system. The MIS gene was cloned into the pBacPAK8 transfer vector and expressed in BMN1 cells, resulting in a ~70 kDa protein that exhibited strong immunoreactivity with anti-human MIS monoclonal antibodies. These findings confirm the expression, molecular identity, and antigenic properties of the recombinant protein. To our knowledge, this is the first report demonstrating MIS expression in B. mori cells using this approach. Beyond cell-based production, the recombinant baculovirus generated in this study can be used to infect silkworm larvae, enabling large-scale, low-cost in vivo expression. This dual platform—combining BMN1 cells and larval bioreactors—provides a flexible and economically efficient strategy for recombinant protein manufacturing.
Overall, this system offers a valuable alternative to traditional mammalian or microbial expression methods, particularly for structurally complex human proteins such as MIS. The platform holds strong potential for future applications in biomedical research, diagnostic assay development, and therapeutic protein production.
Acknowledgements
This work was supported by the Budgetary Program of Basic Scientific Research, Academy of Sciences of the Republic of Uzbekistan (AS RUz).
Data Availability Statement
The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.
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