Use Of Recombinant Proteins Research Articles

Role of Recombinant Proteins for Treating Rheumatoid Arthritis.

Rheumatoid Arthritis (RA) is an autoimmune disease and chronic inflammatory disorder that affects joints and causes inflammation, pain, stiffness, and eventually progressive joint destruction. Approximately 1% of the world's population is estimated to suffer from RA, and if this disease is left untreated, it can lead to severe disability. Despite all the efforts and advances made by professionals in the field, there is currently no definitive treatment for RA, and most treatment strategies are aimed at relieving symptoms and improving patients' quality of life. One of the most promising current approaches is the use of recombinant proteins that target specific signaling pathways involved in the development of RA to alleviate symptoms and slow the progression of the disease. This article discusses the genetic and immunological factors that influence the development of RA, recombinant proteins, methods of using these proteins, approved drugs, and side effects associated with treating RA.

Recombinant Acetylcholine Receptor Immunization Induces a Robust Model of Experimental Autoimmune Myasthenia Gravis in Mice.

Myasthenia gravis (MG) is a prototypical autoimmune disease of the neuromuscular junction (NMJ). The study of the underlying pathophysiology has provided novel insights into the interplay of autoantibodies and complement-mediated tissue damage. Experimental autoimmune myasthenia gravis (EAMG) emerged as a valuable animal model, designed to gain further insight and to test novel therapeutic approaches for MG. However, the availability of native acetylcholine receptor (AChR) protein is limited favouring the use of recombinant proteins. To provide a simplified platform for the study of MG, we established a model of EAMG using a recombinant protein containing the immunogenic sequence of AChR in mice. This model recapitulates key features of EAMG, including fatigable muscle weakness, the presence of anti-AChR-antibodies, and engagement of the NMJ by complement and a reduced NMJ density. Further characterization of this model demonstrated a prominent B cell immunopathology supported by T follicular helper cells. Taken together, the herein-presented EAMG model may be a valuable tool for the study of MG pathophysiology and the pre-clinical testing of therapeutic applications.

Biophysical and biochemical characterization of a recombinant Lyme disease vaccine antigen, CspZ-YA

Lyme disease, caused by Lyme Borrelia spirochetes, is the most common vector-borne illness in the United States. Despite its global significance, with an estimated 14.5 % seroprevalence, there is currently no licensed vaccine. Previously, we demonstrated that CspZ-YA protein conferred protection against Lyme Borrelia infection, making it a promising vaccine candidate. However, such a protein was tagged with hexahistidine, and thus not preferred for vaccine development; furthermore, the formulation to stabilize the protein was understudied. In this work, we developed a two-step purification process for tag-free E. coli-expressed recombinant CspZ-YA. We further utilized various bioassays to analyze the protein and determine the suitable buffer system for long-term storage and formulation as a vaccine immunogen. The results indicated that a buffer with a pH between 6.5 and 8.5 stabilized CspZ-YA by reducing its surface hydrophobicity and colloidal interactions. Additionally, low pH values induced a change in local spatial conformation and resulted in a decrease in α-helix content. Lastly, an optimal salinity of 22–400 mM at pH 7.5 was found to be important for its stability. Collectively, this study provides a fundamental biochemical and biophysical understanding and insights into the ideal stabilizing conditions to produce CspZ-YA recombinant protein for use in vaccine formulation and development.

Use of recombinant proteins for the diagnosis and prevention of Mycoplasma bovis: a systematic review.

Mycoplasma bovis is a highly contagious pathogen that causes various diseases in herd animals, negatively impacting reproduction, production, and milk yield. Effective diagnostic methods and vaccine development are critical for controlling M. bovis outbreaks. This systematic review aimed to evaluate diagnostic alternatives and vaccine compounds based on recombinant proteins. Following the PRISMA protocol, a systematic search was conducted in the SciELO, PubMed, and CAPES Periodicals Portal databases. Inclusion criteria included studies published between 2008 and 2023 that involved (1) the use of recombinant proteins for M. bovis identification or vaccine production, (2) biological samples, (3) availability in the selected databases, (4) in vitro or in vivo experimental designs, and (5) English-language publications. Ten of the initial 53 studies screened met the inclusion criteria. Of these, four studies focused on diagnostic approaches and six on vaccine development. Diagnostic studies predominantly used an indirect enzyme-linked immunosorbent assay (ELISA) with recombinant proteins, achieving over 90% sensitivity and specificity in detecting M. bovis infections. In contrast, the development of recombinant vaccines has shown limited success, with challenges in identifying effective adjuvants and optimizing conditions for protective immunity. While recombinant protein-based diagnostics have proven effective, developing a successful vaccine against M. bovis remains elusive. Further research is necessary to refine vaccine formulations, including selecting suitable adjuvants and challenge models to enhance protective efficacy against M. bovis infections.

Heterologous expression of recombinant proteins and their derivatives used as carriers for conjugate vaccines

The carrier proteins that provide an effective and long-term immune response to weak antigens has become a real breakthrough in disease prevention, making it available to a wider range of patients and making it possible to obtain reliable vaccines against a variety of pathogens. Currently, research is continuing both to identify new peptides, proteins, and their complexes potentially suitable for use as carriers, and to develop new methods for isolation, purification and conjugation of already known and well-proven proteins. The use of recombinant proteins has a number of advantages over isolation from natural sources, such as simpler cultivation of the host organism, the possibility of modifying the genetic construction, the use of numerous promoter variants, signal sequences, and other regulatory elements. This review is devoted to the methods of obtaining both traditional and new recombinant proteins and their derivatives being used or potentially suitable for use as carrier proteins for conjugate vaccines.

Heterologous Expression of Recombinant Proteins and Their Derivatives Used as Carriers for Conjugate Vaccines.

Carrier proteins that provide an effective and long-term immune response to weak antigens has become a real breakthrough in the disease prevention, making it available to a wider range of patients and making it possible to obtain reliable vaccines against a variety of pathogens. Currently, research is continuing both to identify new peptides, proteins, and their complexes potentially suitable for use as carriers, and to develop new methods for isolation, purification, and conjugation of already known and well-established proteins. The use of recombinant proteins has a number of advantages over isolation from natural sources, such as simpler cultivation of the host organism, the possibility of modifying genetic constructs, use of numerous promoter variants, signal sequences, and other regulatory elements. This review is devoted to the methods of obtaining both traditional and new recombinant proteins and their derivatives already being used or potentially suitable for use as carrier proteins in conjugate vaccines.

A Cost-Effective Immobilization Method for MBP Fusion Proteins on Microtiter Plates Using a Gelatinized Starch-Agarose Mixture and Its Application for Convenient Protein-Protein Interaction Analysis.

The detection and quantification of protein-protein interactions (PPIs) is a crucial technique that often involves the use of recombinant proteins with fusion protein tags, such as maltose-binding protein (MBP) and glutathione-S-transferase (GST). In this study, we improved the cohesive and sticky properties of gelatinized starch by supplementing it with agarose, resulting in a harder gel that could coat the bottom of a microtiter plate. The resulting gelatinized starch/agarose mixture allowed for the efficient immobilization of MBP-tagged proteins on the coated plates, enabling the use of indirect ELISA-like PPI assays. By using the enzymatic activity of GST as an indicator, we succeeded in determining the dissociation constants between MBP-tagged and GST-tagged proteins on 96-well microtiter plates and a microplate reader without any expensive specialized equipment.

Notch and Wnt Signaling Modulation to Enhance DPSC Stemness and Therapeutic Potential.

The Dental Pulp of permanent human teeth is home to stem cells with remarkable multilineage differentiation ability: human Dental Pulp Stem Cells (DPSCs). These cells display a very notorious expression of pluripotency core factors, and the ability to give rise to mature cell lineages belonging to the three embryonic layers. For these reasons, several researchers in the field have long considered human DPSCs as pluripotent-like cells. Notably, some signaling pathways such as Notch and Wnt contribute to maintaining the stemness of these cells through a complex network involving metabolic and epigenetic regulatory mechanisms. The use of recombinant proteins and selective pharmacological modulators of Notch and Wnt pathways, together with serum-free media and appropriate scaffolds that allow the maintenance of the non-differentiated state of hDPSC cultures could be an interesting approach to optimize the potency of these stem cells, without a need for genetic modification. In this review, we describe and integrate findings that shed light on the mechanisms responsible for stemness maintenance of hDPSCs, and how these are regulated by Notch/Wnt activation, drawing some interesting parallelisms with pluripotent stem cells. We summarize previous work on the stem cell field that includes interactions between epigenetics, metabolic regulations, and pluripotency core factor expression in hDPSCs and other stem cell types.

Combining fluorescence and electrochemical imaging of chromaffin granule fusion with supported bilayers.

The main barrier to a full understanding of the detailed molecular conformational changes leading to fusion pore formation is the lack of a technique that allows to experimentally measure simultaneously and with (sub-)millisecond time resolution specific conformational changes in the fusion machinery and the formation of the fusion pore. To overcome this barrier we have developed microfabricated electrochemical detector (ECD) arrays patterned on microscope coverslips on which a chromaffin cell can be placed such that the cell surface in contact with the coverslip can simultaneously be imaged in total internal reflection fluorescence (TIRF) excitation mode while the released catecholamine molecules are oxidized by the ECDs producing amperometric oxidation current spikes. The amperometric spikes indicate not only the time of fusion with <1ms precision but also the location of the release event. Using SNAP25-based FRET probes we demonstrated a conformational change in the SNARE complex preceding fusion pore formation. However, the fluorescent protein (FP) based tags, are rather bulky and it is thus not clear what the conformational change exactly is. For a clear interpretation, the use of localized small labels will be needed. Labeling of specific residues requires the use of recombinant proteins in a reconstituted system. We therefore develop a technology where labeled recombinant SNARE proteins will be incorporated into a supported membrane that covers ECD arrays such that TIR-FRET imaging of conformational changes can be performed simultaneously with electrochemical detection of individual fusion events of dense core vesicles with the supported membrane. FRAP measurements show that a fluid membrane is indeed formed on the ECDs and docking and fusion of membrane labeled bovine chromaffin granules could be observed. Supported by NINDS (NIH) R21 NS118319.

Recombinant vaccine candidates with integrated adjuvants provide stimulation of an effective immune response against bacterial infections

The use of recombinant proteins as vaccine preparations is limited by their weak immunogenicity, which can be enhanced by the use of adjuvants, the development of which is an important and urgent problem of modern vaccinology. Significantly, adjuvants as additives to vaccine preparations are of concern to clinicians. From this point of view, the idea of including an internal adjuvant into the structure of a recombinant protein molecule is of undoubted interest. Previously, we synthesized and studied two recombinant vaccine preparations specific for S. agalactiae (Su4) and S. pneumoniae (PSPF). Each of them was a tandem of immunogenic bacterial surface proteins in combination with an additional adjuvant site. The amino acid sequence identical to flagellin acted as an internal adjuvant. In this work, we investigated the possibility of additional enhancement of the body’s immune response to immunization with recombinant Su4 and PSPF proteins due to the simultaneous administration of an external adjuvant, carboxymethylchitosan or Imject Alum.Studies have shown that the additional introduction of these adjuvants into the composition of the vaccine preparation did not affect the immunogenicity of the Su4 and PSPF proteins, which included the internal adjuvant flagellin. The protective efficacy of the immune response to all immunization options was comparable.Thus, the inclusion of a flagellin insert as an internal adjuvant into the composition of recombinant proteins ensures the development of the highest possible level of the immune response and its protective efficacy against the corresponding pathogens of a bacterial infection.

Novel Functional Peptide for Next-Generation Vital Pulp Therapy

Although vital pulp therapy should be performed by promoting the wound-healing capacity of dental pulp, existing pulp-capping materials were not developed with a focus on the pulpal repair process. In previous investigations of wound healing in dental pulp, we found that organic dentin matrix components (DMCs) were degraded by matrix metalloproteinase-20, and DMC degradation products containing protein S100A7 (S100A7) and protein S100A8 (S100A8) promoted the pulpal wound-healing process. However, the direct use of recombinant proteins as pulp-capping materials may cause clinical problems or lead to high medical costs. Thus, we hypothesized that functional peptides derived from recombinant proteins could solve the problems associated with direct use of such proteins. In this study, we identified functional peptides derived from the protein S100 family and investigated their effects on dental pulp tissue. We first performed amino acid sequence alignments of protein S100 family members from several mammalian sources, then identified candidate peptides. Next, we used a peptide array method that involved human dental pulp stem cells (hDPSCs) to evaluate the mineralization-inducing ability of each peptide. Our results supported the selection of 4 candidate functional peptides derived from proteins S100A8 and S100A9. Direct pulp-capping experiments in a rat model demonstrated that 1 S100A8-derived peptide induced greater tertiary dentin formation compared with the other peptides. To investigate the mechanism underlying this induction effect, we performed liquid chromatography–tandem mass spectrometry analysis using hDPSCs and the S100A8-derived peptide; the results suggested that this peptide promotes tertiary dentin formation by inhibiting inflammatory responses. In addition, this peptide was located in a hairpin region on the surface of S100A8 and could function by direct interaction with other molecules. In summary, this study demonstrated that a S100A8-derived functional peptide promoted wound healing in dental pulp; our findings provide insights for the development of next-generation biological vital pulp therapies.

PHYSIOLOGICAL STATE AND PRODUCTIVITY OF CALVES FED GOAT MILK CONTAINING RECOMBINANT HUMAN LACTOFERRIN

The use of recombinant proteins in diets has a positive effect on animal health, promotes intestine recovery and the creation of beneficial microbiota in the gastrointestinal tract. The article contains the results of research in which the effect of &#x0D; frozenthawed milk of goats producing recombinant lactoferrin on hematological parameters and productivity of calves at the age of 1-30 days was studied. Morpho-biochemical blood parameters were found to be within the physiological norms. The use of goat milk containing recombinant human lactoferrin in the diets of calves in the amount of 0.44-0.66 kg/head per day allows to increase the average daily live weight gain by 10,1-11,0% and reduce feed costs by 7.5- 7.7%.

Identification, molecular characterization, and in silico structural analysis of larval salivary glands Netrin-A as a potent biomarker from Lucilia sericata (Diptera: Calliphoridae)

The greenbottle blowfly Lucilia sericata (L. sericata) is increasingly used in larval therapy of chronic wounds. Netrins as bifunctional proteins are in the superfamily of Laminins secreted from larval salivary glands. The Netrin protein has a significant instructive role in axon guidance, causing neuronal outgrowth, angiogenesis, and cell migration. It seems to be crucial in wound healing and acts as a potential biomarker in diagnosing some clinical diseases. This survey aimed to identify molecular features and analyze in silico structural configuration of Netrin-A in L. sericata larvae. The larvae were reared under standard maggotarium conditions. The nucleic acid sequence of L. sericata Netrin-A (LSN-A) was then identified using rapid amplification of circular DNA ends (RACE) and rapid amplification of genomic ends (RAGE). Parts of the Netrin-A gene, including the middle, 3'-, and 5'-ends, were identified, TA cloned in pTG19 plasmid, and transferred into DH5ɑ Escherichia coli. Each part was sequenced and assembled using SeqMan software. This gene structure was further subjected to in silico analysis. The DNA of LSN-A was identified to be 2407bp, while its mRNA sequence was recognized as 2115bp by Oligo0.7 software. It translated the Netrin-A protein with 704 amino acid residues. Its estimated molecular weight was 78.6kDa. Sequencing of this fragment and its BLAST analysis revealed laminin-based high (95%) similarity with the mRNA sequence of Lucilia cuprina Netrin-A. The 3-D structure of Netrin-A drawn by SWISS-MODEL exhibited its partial resemblance to the reference molecule Netrin-1 of Homo sapiens. This study supports the molecular and structural analyses of LSN-A protein, which could lead to wound treatment. Ultimately, it can be an effective candidate to ameliorate injury. Our next attempt is to produce LSN-A recombinant protein for use in biomedical sciences.

Immune Evaluation of Avian Influenza Virus HAr Protein Expressed in Dunaliella salina in the Mucosa of Chicken.

Avian influenza (AI) is a serious threat to the poultry industry worldwide. Currently, vaccination efforts are based on inactivated, live attenuated, and recombinant vaccines, where the principal focus is on the type of virus hemagglutinin (HA), and the proposed use of recombinant proteins of AI virus (AIV). The use of antigens produced in microalgae is a novel strategy for the induction of an immune response in the mucosal tissue. The capacity of the immune system in poultry, particularly in mucosa, plays an important role in the defense against pathogens. This system depends on a complex relationship between specialized cells and soluble factors, which confer protection against pathogens. Primary lymphoid organs (PLO), as well as lymphocytic aggregates (LA) such as the Harderian gland (HG) and mucosa-associated lymphoid tissue (MALT), actively participate in a local immune response which is mainly secretory IgA (S-IgA). This study demonstrates the usefulness of subunit antigens for the induction of a local and systemic immune response in poultry via ocular application. These findings suggest that a complex protein such as HAr from AIV (H5N2) can successfully induce increased local production of S-IgA and a specific systemic immune response in chickens.

Use of recombinant proteins in the diagnosis of sexually transmitted infections: a systematic review and meta-analysis

The present systematic review and meta-analysis aimed to investigate the use of recombinant proteins to diagnose STIs and evaluate the performance of the tests identified. The databases, SciELO, PubMed, CAPES Journal Portal, and LILACS, were searched according to the PRISMA protocol. A study was considered eligible if it met previously defined criteria. The risk of study bias was assessed according to the QUADAS-2 protocol. The meta-analysis was performed based on the random-effects model, and heterogeneity was quantified using the I² statistic. A total of 1,355 studies were selected, 30 of which were relevant to the following: human immunodeficiency virus (12/30), Treponema pallidum (10/30), Chlamydia trachomatis (3/30), herpes simplex virus (HSV, 2/30), Trichomonas vaginalis (2/30), and HSV/cytomegalovirus (1/30). The main serological tests were ELISA and immunochromatography. The risk of bias was low for most included studies. According to each microorganism, the meta-analysis revealed satisfactory sensitivity and specificity for the analyzed tests. The findings reinforce the relevance of diagnostic tests based on recombinant proteins as viable alternatives for production and inclusion in clinical practice. PROSPERO registration number: CRD42020206331.

Evaluation of two heterologous recombinant antigens for the serological diagnosis of human polycystic echinococcosis.

Polycystic echinococcosis (PE) is a zoonosis endemic in the Neotropical region of the Americas. It is caused by the larval stage of the cestode Echinococcus vogeli, which develops as harmful cysts that slowly grow in the liver, lungs and other organs of humans and other host species. Human PE diagnosis is usually based on clinical and epidemiological aspects and imaging techniques, often requiring confirmation by immunological assays. The currently available serological tests for detecting antibodies against Echinococcus spp. are mostly based on complex, variable and poorly characterized mixtures of native parasite antigens, which impairs specificity and/or sensitivity. In this scenario, the evaluation of well-characterized alternative antigens is urgently needed for the improvement of PE diagnosis. Here, two subunits (AgB8/1 and AgB8/2) of the major secretory antigen from Echinococcus granulosus (antigen B (AgB)), of diagnostic value for cystic echinococcosis, were validated for PE diagnosis. These antigens, produced as pure recombinant proteins (rAgB8/1 and rAgB8/2) in Escherichia coli, allowed detecting specific immunoglobulin G antibodies in sera from PE patients in an enzyme-linked immunosorbent assay, with sensitivities of 83.72% and 81.40%, respectively, and specificities of 83.12% and 80.09%, respectively. The use of recombinant proteins overcomes difficulties to obtain parasite material and reduced non-specific reactions and costs. Our results demonstrated reproducibility and accuracy high enough to be considered valid according to the acceptance criteria for Food and Drug Administration assay validation. This qualifies rAgB8/1 and rAgB8/2 as potential substitutes for the currently used parasite crude or partially purified antigens.

Fifty years of Bryology in Mexico

Background: Mexican botanists were not involved in bryophyte research fifty years ago; only four small floristic contributions were published between 1942-1958.&#x0D; Questions: What has been learned in the last fifty years? How many bryophytes are there in Mexico? What are the contributions by Mexican scientists?&#x0D; Studied species: Bryophyte flora.&#x0D; Study site and dates: Mexico, between 1942-2021.&#x0D; Methods: Bibliographic compilations were used to revise the status of bryophyte research in Mexico. Data for the last fifty years cited there and in an updated version of Latmoss served to determine the current knowledge of Mexican bryophytes as contributed by Mexican scientists. No thesis research was considered unless published in a scientific journal.&#x0D; Results: There are 16 species of Anthocerotophyta, ca. 600 of Marchantiophyta, and 997 Bryophyta in Mexico. At least seven phytogeographic elements are represented: Northern, Meso-American, Caribbean, Southern, Wide distribution, and Endemic. Highlights of Mexican research include the discovery of Hypnodontopsis sp., a Miocene amber fossil from Chiapas, identification of heavy metals deposits in urban mosses in Mexico City and Toluca, determination of chloroplast and mitochondrial genomes of Pseudocrossidium replicatum , and the potential use of recombinant proteins from Physcomitrella patens .&#x0D; Conclusions: Taxonomic and floristic studies should be continued along with the bryological exploration of the country. Conservation is urgent, but studies of drought tolerance, air pollution, climate change, and potential uses in medicine require support and collaboration from other scientists.

Serologic Cross-Reactivity of SARS-CoV-2 with Endemic and Seasonal Betacoronaviruses.

In order to properly understand the spread of SARS-CoV-2 infection and development of humoral immunity, researchers have evaluated the presence of serum antibodies of people worldwide experiencing the pandemic. These studies rely on the use of recombinant proteins from the viral genome in order to identify serum antibodies that recognize SARS-CoV-2 epitopes. Here, we discuss the cross-reactivity potential of SARS-CoV-2 antibodies with the full spike proteins of four other betacoronaviruses that cause disease in humans, MERS-CoV, SARS-CoV, HCoV-OC43, and HCoV-HKU1. Using enzyme-linked immunosorbent assays (ELISAs), we detected the potential cross-reactivity of antibodies against SARS-CoV-2 towards the four other coronaviruses, with the strongest cross-recognition between SARS-CoV-2 and SARS /MERS-CoV antibodies, as expected based on sequence homology of their respective spike proteins. Further analysis of cross-reactivity could provide informative data that could lead to intelligently designed pan-coronavirus therapeutics or vaccines.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10875-021-00997-6.

Mutation of cysteine residues increases heterologous expression of peach expansin in the methylotrophic yeast &lt;i&gt;Pichia pastoris&lt;/i&gt;

The study of Carbohydrate-Active enZymes (CAZymes) associated with plant cell wall metabolism is important for elucidating the developmental mechanisms of plants and also for the utilization of plants as a biomass resource. The use of recombinant proteins is common in this context, but heterologous expression of plant proteins is particularly difficult, in part because the presence of many cysteine residues promotes denaturation, aggregation and/or protein misfolding. In this study, we evaluated two phenotypes of methylotrophic yeast Pichia pastoris as expression hosts for expansin from peach (Prunus persica (L.) Batsch, PpEXP1), which is one of the most challenging targets for heterologous expression. cDNAs encoding wild-type expansin (PpEXP1_WT) and a mutant in which all cysteine residues were replaced with serine (PpEXP1_CS) were each inserted into expression vectors, and the protein expression levels were compared. The total amount of secreted protein in PpEXP1_WT culture was approximately twice that of PpEXP1_CS. However, the amounts of recombinant expansin were 0.58 and 4.3 mg l-1, corresponding to 0.18% and 2.37% of total expressed protein, respectively. This 13-fold increase in production of the mutant in P. pastoris indicates that the replacement of cysteine residues stabilizes recombinant PpEXP1.

Gene flow from transgenic soybean, developed to obtain recombinant proteins for use in the skin care industry, to non-transgenic soybean

Soybean has been recognized as a useful platform for heterologous protein production. This study compared the pollen characteristics of transgenic and non-transgenic soybean and investigated the rate of gene flow from transgenic soybean events, developed to obtain recombinant proteins (such as human epidermal growth factor, insulin-like growth factor 1, or thioredoxin) for use in the skin care industry, to non-transgenic soybean under field conditions, and determined the distance at which gene flow could occur. The lack of significant differences in pollen grain size, viability and pollen germination rates between transgenic and non-transgenic cultivars indicates that the overexpression of transgenes did not alter pollen characteristics in soybean. The highest rates of gene flow from the three transgenic soybean events to non-transgenic soybean ranged from 0.22 to 0.46% at the closest distance (0.5 m). Gene flow was observed up to 13.1 m from the transgenic plots. Our data fell within the ranges reported in the literature and indicate that an isolation distance greater than at least 13 m from transgenic soybean is required to prevent within-crop gene flow in soybean. As the potential markets for transgenic crops as a recombinant protein factory increase, gene flow from transgenic to non-transgenic conventional crops will become a key decision factor for policy makers during the approval process of transgenic crops. Our study may provide useful baseline data for the prevention of transgenic soybean seed contamination caused by transgene flow.