Expression Of Recombinant Antibodies Research Articles

In planta expression of specific single chain fragment antibody (scFv) against nucleocapsid protein of fig mosaic virus (FMV)

Fig mosaic virus (FMV) is recognized as the main viral agent associated with the mosaic disease (MD) of fig trees (Ficus carica). Due to its worldwide occurrence, FMV represents the most significant global threat to the production of fig fruit. A disease management strategy against the MD in fig orchards has never been effective; and therefore, expression of recombinant antibody in plant cells could provide an alternative approach to suppress FMV infections. In this study we focused on expressing a specific recombinant antibody, a single-chain variable fragment (scFv), targeting the nucleocapsid protein (NP) of FMV in planta. To accomplish this objective, we inserted the scFv gene into a plant expression vector and conducted transient expression in leaves of Nicotiana tabacum cv. Samson plants. The construct was transiently expressed in tobacco plants by agroinfiltration, and antibody of the anticipated size was detected by immunoblotting. The produced plantibody was then assessed for specificity using ELISA and confirmed by Western blot analysis. In this study, the plantibody developed against FMV could be considered as a potential countermeasure to the infection by conferring resistance to MD.

Duplication of a chromosome 2 segment in production CHO cell lines correlates with age-related growth improvement.

Monitoring the stability of recombinant Chinese Hamster Ovary (CHO) cell lines is essential to ensure the selection of production cell lines suitable for biomanufacturing. It has been frequently observed that recombinant CHO cell lines develop phenotypic changes upon aging, such as accelerated cell growth in late generation cultures. However, the mechanism responsible for age-correlated changes is poorly understood. In this study, we investigated the molecular mechanisms underlying the age-correlated cell growth improvement in Pfizer's platform fed-batch production process, by examining multiple cell lines derived from different CHO expression systems, expressing a variety of monoclonal antibodies (mAbs). Comprehensive whole-genome resequencing analysis revealed duplication of a continuous 50.2 Mbp segment in chromosome 2 (Chr2) specific to clones that showed age-correlated growth change as compared to clones that did not exhibit age-correlated growth change. Moreover, such age- and growth-related Chr2 duplication was independent of the presence or type of recombinant monoclonal antibody expression. When we compared transcriptome profiles from low-growth and high-growth cell lines, we found that>95% of the genes overexpressed in high-growth cell lines were in the duplicated Chr2 segment. To the best of our knowledge, this is the first report of large genomic duplication, specific to Chr2, being associated with age-correlated growth change. Investigation of the cause-and-effect relationship between the genes identified in the duplicated regions and age-correlated growth change is underway. We are confident that this effort will lead to improved cell line screening and targeted rational cell line engineering efforts to develop cell lines with improved stability performance.

Rapid generation of human recombinant monoclonal antibodies from antibody-secreting cells using ferrofluid-based technology.

Monoclonal antibodies (mAbs) are one of the most important classes of biologics with high therapeutic and diagnostic value, but traditional methods for mAbs generation, such as hybridoma screening and phage display, have limitations, including low efficiency and loss of natural chain pairing. To overcome these challenges, novel single B cell antibody technologies have emerged, but they also have limitations such as in vitro differentiation of memory B cells and expensive cell sorters. In this study, we present a rapid and efficient workflow for obtaining human recombinant monoclonal antibodies directly from single antigen-specific antibody secreting cells (ASCs) in the peripheral blood of convalescent COVID-19 patients using ferrofluid technology. This process allows the identification and expression of recombinant antigen-specific mAbs in less than 10 days, using RT-PCR to generate linear Ig heavy and light chain gene expression cassettes, called "minigenes", for rapid expression of recombinant antibodies without cloning procedures. This approach has several advantages. First, it saves time and resources by eliminating the need for in vitro differentiation. It also allows individual antigen-specific ASCs to be screened for effector function prior to recombinant antibody cloning, enabling the selection of mAbs with desired characteristics and functional activity. In addition, the method allows comprehensive analysis of variable region repertoires in combination with functional assays to evaluate the specificity and function of the generated antigen-specific antibodies. Our approach, which rapidly generates recombinant monoclonal antibodies from single antigen-specific ASCs, could help to identify functional antibodies and deepen our understanding of antibody dynamics in the immune response through combined antibody repertoire sequence analysis and functional reactivity testing.

Production of novel recombinant anti-EpCAM antibody as targeted therapy for breast cancer

BackgroundThe utilization of monoclonal antibodies (moAbs), an issue correlated with the biopharmaceutical professions, is developing and maturing. Coordinated with this conception, we produced the appealingly modeled anti-EpCAM scFv for breast cancer tumors. MethodsAfterward cloning and expression of recombinant antibody in Escherichia coli bacteria, the correctness of the desired antibody was checked by western blotting. Flow cytometry was utilized to determine the capacity of the recombinant antibody to append to the desired receptors in the malignant breast cancer (BC)cell line. The recombinant antibody (anti-EpCAM scFv) was examined for preclinical efficacy in reducing tumor growth, angiogenesis, and invasiveness (in vitro- in vivo). FindingsA target antibody-mediated attenuation of migration and invasion in the examined cancer cell lines was substantiated (P-value < 0.05). Grafted tumors from breast cancer in mice indicated significant and compelling suppression of tumor growth and decrement in blood supply in reaction to the recombinant anti-EpCAM intervention. Evaluations of immunohistochemical and histopathological findings revealed an enhanced response rate to the treatment. ConclusionThe desired anti-EpCAM scFv can be a therapeutic tool to reduce invasion and proliferation in malignant breast cancer.

Strategies and Considerations for Improving Recombinant Antibody Production and Quality in Chinese Hamster Ovary Cells.

Recombinant antibodies are rapidly developing therapeutic agents; approximately 40 novel antibody molecules enter clinical trials each year, most of which are produced from Chinese hamster ovary (CHO) cells. However, one of the major bottlenecks restricting the development of antibody drugs is how to perform high-level expression and production of recombinant antibodies. The high-efficiency expression and quality of recombinant antibodies in CHO cells is determined by multiple factors. This review provides a comprehensive overview of several state-of-the-art approaches, such as optimization of gene sequence of antibody, construction and optimization of high-efficiency expression vector, using antibody expression system, transformation of host cell lines, and glycosylation modification. Finally, the authors discuss the potential of large-scale production of recombinant antibodies and development of culture processes for biopharmaceutical manufacturing in the future.

Defective Early B Cell Tolerance Checkpoints in Patients With Systemic Sclerosis Allow the Production of Self Antigen-Specific Clones.

Early selection steps preventing autoreactive naive B cell production are often impaired in patients with autoimmune diseases, but central and peripheral B cell tolerance checkpoints have not been assessed in patients with systemic sclerosis (SSc). This study was undertaken to characterize early B cell tolerance checkpoints in patients with SSc. Using an in vitro polymerase chain reaction-based approach that allows the expression of recombinant antibodies cloned from single B cells, we tested the reactivity of antibodies expressed by 212 CD19+CD21low CD10+IgMhigh CD27- new emigrant/transitional B cells and 190 CD19+CD21+CD10-IgM+CD27- mature naive B cells from 10 patients with SSc. Compared to serum from healthy donors, serum from patients with SSc displayed elevated proportions of polyreactive and antinuclear-reactive new emigrant/transitional B cells that recognize topoisomerase I, suggesting that defective central B cell tolerance contributes to the production of serum autoantibodies characteristic of the disease. Frequencies of autoreactive mature naive B cells were also significantly increased in SSc patients compared to healthy donors, thus indicating that a peripheral B cell tolerance checkpoint may be impaired in SSc. Defective counterselection of developing autoreactive naive B cells in SSc leads to the production of self antigen-specific B cells that may secrete autoantibodies and allow the formation of immune complexes, which promote fibrosis in SSc.

Profiling the specificity of clonally expanded plasma cells during chronic viral infection by single-cell analysis.

Plasma cells and their secreted antibodies play a central role in the long‐term protection against chronic viral infection. However, due to experimental limitations, a comprehensive description of linked genotypic, phenotypic, and antibody repertoire features of plasma cells (gene expression, clonal frequency, virus specificity, and affinity) has been challenging to obtain. To address this, we performed single‐cell transcriptome and antibody repertoire sequencing of the murine BM plasma cell population following chronic lymphocytic choriomeningitis virus infection. Our single‐cell sequencing approach recovered full‐length and paired heavy‐ and light‐chain sequence information for thousands of plasma cells and enabled us to perform recombinant antibody expression and specificity screening. Antibody repertoire analysis revealed that, relative to protein immunization, chronic infection led to increased levels of clonal expansion, class‐switching, and somatic variants. Furthermore, antibodies from the highly expanded and class‐switched (IgG) plasma cells were found to be specific for multiple viral antigens and a subset of clones exhibited cross‐reactivity to nonviral and autoantigens. Integrating single‐cell transcriptome data with antibody specificity suggested that plasma cell transcriptional phenotype was correlated to viral antigen specificity. Our findings demonstrate that chronic viral infection can induce and sustain plasma cell clonal expansion, combined with significant somatic hypermutation, and can generate cross‐reactive antibodies.

Effects of ubiquitous chromatin opening element (UCOE) on recombinant anti-TNFα antibody production and expression stability in CHO-DG44 cells.

To date, the production of antibodies (mAbs) usually faces the risks of transgene expression reduction and instability, especially after long-time culture. The inclusion of ubiquitous chromatin opening element (UCOE) into expression vectors was reported to enhance protein production and maintain transgene expression stability in CHO cell lines. Thus, we investigate the effects of UCOE on recombinant monoclonal anti-TNFα antibody (mAbTNFα) production and expression stability in CHO-DG44 cells. In our study, non-UCOE and UCOE-based vectors encoding mAbTNFα were constructed and introduced into the CHO-DG44 cells. Cell pools and single-cell clones were obtained by selecting transfected cells with G418, amplifying them by treatment with methotrexate (MTX), and isolating them by limiting dilution. The effects of UCOE on mAb production and stable transgene expression in transfected cells were analyzed via the correlation between mAb yields and mRNA expression level variations, and gene copy number changes. The UCOE pool exhibited higher mAb yield compared to non-UCOE pool. The UCOE was associated with higher transgene transcriptional activity, leading to improvement of mAb production after MTX-mediated gene amplification. The incorporation of UCOE generated cells allowed isolation of greater numbers of positive clones with higher expression. Despite the slightly decreased mAb yield, UCOE clones still retain stable long-term expression in the absence of selective pressure, which was explained by the loss of transgene copies rather than due to the decline of transcriptional activity. In addition, the purified mAb had primary chemical and biological characteristics similar to those of adalimumab. The results showed that the incorporation of UCOE within vectors provides significant advantages in the generation of high-producing clones, enhancement of mAb production, and improvement of gene expression stability.

Production of Monoclonal Antibodies Targeting Aminopeptidase N in the Porcine Intestinal Mucosal Epithelium.

Porcine aminopeptidase N (APN), a membrane-bound metallopeptidase abundantly present in small intestinal mucosa, can initiate a mucosal immune response without any interference such as low protein expression, enzyme inactivity, or structural changes. This makes APN an attractive candidate in the development of vaccines that selectively target the mucosal epithelium. Previous studies have shown that APN is a receptor protein for both enterotoxigenic Escherichia coli (E. coli) F4 and transmissible gastroenteritis virus. Thus, APN shows promise in the development of antibody-drug conjugates or novel vaccines based on APN-specific antibodies. In this study, we compared production of APN-specific monoclonal antibodies (mAbs) using traditional hybridoma technology and recombinant antibody expression method. We also established a stably transfected Chinese hamster ovary (CHO) cell line using pIRES2-ZSGreen1-rAbs-APN and an E. coli expression BL21(DE3) strain harboring the pET28a (+)-rAbs-APN vector. The results show that antibodies expressed in pIRES2-ZSGreen1-rAbs-APN-CHO cells and mAbs produced using hybridomas could recognize and bind to the APN protein. This provides the basis for further elucidation of the APN receptor function for the development of therapeutics targeting different APN-specific epitopes.

Increasing transgene expression and stability in recombinant CHO cells with DNA methyltransferase Dnmt3b gene knockout via CRISPR/Cas9

Chinese hamster ovary (CHO) cells are the preferred producers because of their capacity to perform proper protein folding, assembly, and post-translational modifications, similar to human cells. When CHO cells are used to produce recombinant drug proteins, it is critical to maintain stable subculture of transgenic CHO cells and high expression levels of target genes when the recombinant expression seed cell bank is expanded to a large scale bioreactor. However, at present, the instability of recombinant protein expression during prolonged culture is one of the most common bottlenecks in mammalian protein production restricting the development of CHO cell expression system. It is believed that the instability of transgene expression in CHO cells is closely related to DNA methylation. DNA methylation was mainly catalyzed by the de novo methyltransferase Dnmt3a, Dnmt3b and DNA methylsustaining-enzyme Dnmt1. DNA methyltransferase Dnmt3a and 3b play an important role in gene silencing induced by epigenetic modifications such as DNA methylation. It has been found that epigenetic modification mediated by Dnmt3b is more closely related to gene expression regulation. Therefore, in this paper, Dnmt3b gene-deficient CHO cells were established through knock out of the Dnmt3b gene by CRISPR/Cas9 genome editing technology. Furthermore, the stability and efficiency of the expression system based on the Dnmt3b KO CHO cells were evaluated by determining the expression levels of the recombinant proteins. The results indicated that the long-term stability of the green fluorescent reporter gene EGFP in the transfected Dnmt3b KO CHO cells over 30 passages increased regardless of the presence or absence of G418, compared with the normal transfected CHO cells. We also found that the expression of recombinant human-vitronectin(VN) and monoclonal antibodies (mAbs) in the stable cell pools of the transgenic Dnmt3b KO CHO cells were at least 1.6 folds higher than those of the normal CHO cells. Under the condition of not increasing the cost of culture, it is beneficial to the screening of high yielding cells and industrial production. The findings indicated that the Dnmt3b-deficient CHO cells could significantly improve the stability of target gene expression, which provide theoretical significance and application value for developing novel effective and stable CHO cell expression system.

Expression and purification of a novel single-chain diabody (scDb-hERG1/β1) from Pichia pastoris transformants

In the last decades, protein engineering has developed particularly in biotechnology and pharmaceutical field. In particular, the engineered antibody subclass has arisen. The single chain diabody format (scDb), conjugating small size with antigen specificity, offers versatility representing a gold standard for a variety of applications, spacing from research to diagnostics and therapy. Along with such advantages, comes the challenge of optimizing their production, improving expression systems, purification procedures and stability. All such parameters are detrimental for protein production in general and above all for recombinant antibody expression, which has to be fine-tuned, choosing a proper protein-expression host and adjusting expression protocols accordingly. In the present paper, we present data regarding the production and purification of a single chain diabody directed against the macromolecular complex hERG1/β1 integrin. We focus on the expression of clones deriving from the transformation of Pichia pastoris yeast cells. In particular, we compare two different clones arose from two separate transformation processes, demonstrating that both are suitable for proper protein expression. Moreover, we have set up an expression protocol and compared the yields obtained using two purification machines: Akta Pure and Akta Start, with a positive outcome.

Statistical optimization of culture conditions for expression of recombinant humanized anti-EpCAM single-chain antibody using response surface methodology.

Background and purpose:The epithelial cell adhesion molecule (EpCAM), is one of the first cancer- associated markers discovered. Its overexpression in cancer stem cells, epithelial tumors, and circulating tumor cells makes this molecule interesting for targeted cancer therapy. So, in recent years scFv fragments have been developed for EpCAM targeting.Experimental approach:In this study, an scFv against EpCAM extracellular domain (EpEX) derived from 4D5MOC-B humanized mAb was expressed in Escherichia coli k12 strain, and in order to obtain the optimum culture conditions in chemically defined minimal medium, response surface methodology (RSM) was employed. According to the RSM-CCD method, a total of 30 experiments were designed to investigate the effects of various parameters including isopropyl-b-D-thiogalactopyranoside (IPTG) concentration, cell density before induction, post-induction time, and post-induction temperature on anti EpEX-scFv expression level.Findings/Results:At the optimum conditions (induction at cell density 0.8 with 0.8 mM IPTG for 24 h at 37 °C), the recombinant anti EpEX-scFv was produced at a titer of 197.33 μg/mL that was significantly consistent with the prediction of the model.Conclusion and implication:The optimized-culture conditions obtained here for efficient production of anti EpEX-scFv in shake flask cultivation on a chemically defined minimal medium could be applied to large- scale fermentation for the anti EpEX-scFv production.

Rational Design of Single Copy Expression Cassettes in Defined Chromosomal Sites Overcomes Intraclonal Cell-to-Cell Expression Heterogeneity and Ensures Robust Antibody Production.

The expression of endogenous genes as well as transgenes depends on regulatory elements within and surrounding genes as well as their epigenetic modifications. Members of a cloned cell population often show pronounced cell-to-cell heterogeneity with respect to the expression of a certain gene. To investigate the heterogeneity of recombinant protein expression we targeted cassettes into two preselected chromosomal hot-spots in Chinese hamster ovary (CHO) cells. Depending on the gene of interest and the design of the expression cassette, we found strong expression variability that could be reduced by epigenetic modifiers, but not by site-specific recruitment of the modulator dCas9-VPR. In particular, the implementation of ubiquitous chromatin opening elements (UCOEs) reduced cell-to-cell heterogeneity and concomitantly increased expression. The application of this method to recombinant antibody expression confirmed that rational design of cell lines for production of transgenes with predictable and high titers is a promising approach.

Validation of the Production of Antibodies in Different Formats in the HEK 293 Transient Gene Expression System.

Mammalian cells are the most commonly used production system for therapeutic antibodies. Protocols for the expression of recombinant antibodies in HEK293-6E cells in different antibody formats are described in detail. As model, antibodies against Kallikrein-related peptidase 7 (KLK7) were used. KLK7 is a key player in skin homeostasis and represents an emerging target for pharmacological interventions. Potent inhibitors can not only help to elucidate physiological and pathophysiological functions but also serve as a new archetype for the treatment of inflammatory skin disorders. Phage display-derived affinity-matured human anti-KLK7 antibodies were converted to scFv-Fc, IgG, and Fab formats and transiently produced in the mammalian HEK293-6E system. For the production of the corresponding antigen-KLK7-the baculovirus expression vector system (BEVS) and virus-free expression in Hi5 insect cells were used in a comparative approach. The target proteins were isolated by various chromatographic methods in a one- or multistep purification strategy. Ultimately, the interaction between anti-KLK7 and KLK7 was characterized using biolayer interferometry. Here, protocols for the expression of recombinant antibodies in different formats are presented and compared for their specific features. Furthermore, biolayer interferometry (BLI), a fast and high-throughput biophysical analytical technique to evaluate the kinetic binding constant and affinity constant of the different anti-KLK7 antibody formats against Kallikrein-related peptidase 7 is presented.

Создание стабильной высокопродуктивной платформы на основе клеток CHO, адаптированных к химическому и механическому стрессам

Возрастающий спрос на получение рекомбинантных молекул требует создания не только стабильных, но и высокопроизводительных клеточных линий, пригодных для масштабного производства. В настоящей работе предложен дизайн вектора для эффективной и стабильной экспрессии рекомбинатных моноклональных антител в клетках CHO и схема адаптации линии CHO-S (реципиента) к высокой осмоляльности среды в условиях повышенного содержания хлорида натрия (до 100 мМ), лактата натрия (до 170 мМ) и скорости перемешивания в биореакторе до 240 об/мин. Показана эффективность полученной клеточной платформы на примере экспрессии моноклонального антитела IgG2/4k, позволяющей получать до 2,5 г/л продукта без амплификации гена интереса и оптимизации условий культивирования в режиме fed-batch.

Construction, expression and functional analysis of anti-clenbuterol codon-optimized scFv recombinant antibody

The construction, expression and functional analysis of codon-optimized single-chain variable fragment (coscFv) against clenbuterol (CBL) prepared from the Escherichia coli system is described. First, the ionic concentration for coscFv expression was optimized through single-factor experiments. Then, the extraction conditions of inclusion bodies were optimized, and coscFv was affinity-purified. Finally, the functional analysis of coscFv was elucidated by indirect competitive enzyme-linked immunosorbent assay (icELISA) and molecular docking. After optimizing the ionic concentration, the yield of coscFv increased from 21.69% to 23.26%. The molecular weight of coscFv was determined to be approximately 27 kDa according to the SDS-PAGE and Western blot assay. The percentage of coscFv was as high as 43.9% after the inclusion bodies were extracted, washed, and dissolved. Functional analysis indicated that the coscFv recognized CBL, and the 50% inhibition average concentration of CBL (IC50) was 4.22 ± 0.01 (n = 3) ng/mL. The binding site between coscFv and CBL consisted of Asp33H, Met34H, Ser50H, Arg52H, Tyr57H, Leu59H, Asp99H, and Tyr93L. Our study confirms that coscFv can bind with CBL through the key amino acid residues and can be used to sensitively detect CBL.

Development of a new promoter to avoid the silencing of genes in the production of recombinant antibodies in chinese hamster ovary cells

BackgroundThe production of recombinant proteins in mammalian cell lines is one of the most important areas in biopharmaceutical industry. Viral transcriptional promoters are widely used to express recombinant proteins in mammalian cell lines. However, these promoters are susceptible to silencing, thus limiting protein productivity. Some CpG islands can avoid the silencing of housekeeping genes; for that reason, they have been used to increase the production of recombinant genes in cells of animal origin. In this study, we evaluated the CpG island of the promoter region of the β-actin gene of Cricetulus griseous (Chinese hamster), associated to the Cytomegalovirus (CMV) promoter, to increase recombinant antibodies production in Chinese Hamster Ovary (CHO) cells.ResultsWe focused on the non-coding region of CpG island, which we called RegCG. RegCG behaved as a promoter, whose transcriptional activity was mainly commanded by the CAAT and CArG boxes of the proximal promoter. However, the transcription started mainly at the intronic region before the proximal transcription start site. While the CMV promoter was initially more powerful than RegCG, the latter promoter was more resistant to silencing than the CMV promoter in stable cell lines, and its activity was improved when combined with the CMV promoter. Thereby, the chimeric promoter was able to maintain the expression of recombinant antibodies in stable clones for 40 days at an average level 4 times higher than the CMV promoter. Finally, the chimeric promoter showed compatibility with a genetic amplification system by induction with methotrexate in cells deficient in the dihydrofolate reductase gene.ConclusionsWe have generated an efficient synthetic hybrid transcription promoter through the combination of RegCG with CMV, which, in stable cell lines, shows greater activity than when both promoters are used separately. Our chimeric promoter is compatible with a genetic amplification system in CHO DG44 cells and makes possible the generation of stable cell lines with high production of recombinant antibodies. We propose that this promoter can be a good alternative for the generation of clones expressing high amount of recombinant proteins, essential for industrial applications.

Expression of the recombinant single chain variable fragments recognizing blood antigen fused with thioredoxin in &lt;i&gt; Escherichia coli &lt;/i&gt;

The technology of recombinant single chain variable fragments (scFvs) expression has been used in research, diagnosis and treatment of diseases. In the previous study, we studied the expression of a recombinant single chain variable fragment recognizing blood A antigen (antiA-scFv) in E. coli. However, the protein was insoluble form resulting in difficulty for purification, refolding and activity assesment. Here, we present the study on fused expression of the recombinant scFv -specific blood A antigen with thioredoxin (Trx) in the expression vector pET32a (+). The results showed that the Trx/antiA-scFv fusion protein was expressed with molecular weight of 49 kDa in a soluble form reaching 40% of the total recombinant protein. This result facilitates the optimal condition of soluble protein expression, purification and bioactivity determination of the antiA-scFv recombinant antibody.

Advances in antibody drug expression techniques

The 21st century is regarded as the century of biotechnological drugs, among which monoclonal antibodies and their derived targeting drugs have established themselves as the leading modality of biopharmaceutical pharmaceutics for a wide range of indications covering malignant tumors and autoimmune disorders. Since the manufacturing of the first antibody drug from hybridoma cells, the technologies have been intensely studied and there emerged numerous breakthroughs in recombinant cell line establishment, antibody expression and purification, quality control and other related areas. This article summarizes the critical progresses of antibody drugs expression technologies, especially of mammalian cell expression system, Escherichia coli expression system, the transgenic animal reactor and the cell free protein synthesis system, to give a detailed illustration of the recent advances in antibody drugs development.

Cytoplasmic and periplasmic expression of recombinant shark VNAR antibody in Escherichia coli

Shark variable new antigen receptors (VNARs) are known to possess excellent heat-stability, and the long complementarity determining region 3 (CDR3) has permitted it to penetrate into the cleft region of antigens. The number of cysteine (Cys) residues contained within VNAR is greater than in conventional antibodies, entailing disulfide bond formation in both the inter- or intra-loop regions is required for interactions with the target protein antigens. Therefore, the selection of a suitable expression system is important to ensure the solubility and correct folding of functional VNAR protein production. Unlike higher organisms, the machinery for effecting posttranslational modifications of proteins in Escherichia coli (E. coli) are less sophisticated. To overcome this circumstance, a pDSB-28Y vector fusion with DsbA signal peptide was engineered for periplasmic H8VNAR production. Despite the periplasmic proteins showing a lower yield (62 µg/mL) than cytosolic proteins (468 µg/mL) that is obtained from pET-28a vector, it has demonstrated better performance than that of a cytosolic protein in terms of absorbance. However, these readings were still inferior to that of positive control mouse monoclonal antibody (mAb) C1-13 in this experiment. Therefore, further investigation is required to improve the binding affinity of selected recombinant VNAR towards malaria biomarkers.