Increasing CRISPR/Cas9-mediated gene editing efficiency in T7 phage by reducing the escape rate based on insight into the survival mechanism.

Bacteriophages have been used across various fields, and the utilization of CRISPR/Cas-based genome editing technology can accelerate the research and applications of bacteriophages. However, some bacteriophages can escape from the cleavage of Cas protein, such as Cas9, and decrease the efficiency of genome editing. This study focuses on the bacteriophage T7, which is widely utilized but whose mechanism of evading the cleavage of CRISPR/Cas9 has not been elucidated. First, we test the escape rates of T7 phage at different cleavage sites, ranging from 10 -2 to 10 -5. The sequencing results show that DNA point mutations and microhomology-mediated end joining (MMEJ) at the target sites are the main causes. Next, we indicate the existence of the hotspot DNA region of MMEJ and successfully reduce MMEJ events by designing targeted sites that bypass the hotspot DNA region. Moreover, we also knock out the ATP-dependent DNA ligase 1. 3 gene, which may be involved in the MMEJ event, and the frequency of MMEJ at 4. 3 is reduced from 83% to 18%. Finally, the genome editing efficiency in T7 Δ 1. 3 increases from 20% to 100%. This study reveals the mechanism of T7 phage evasion from the cleavage of CRISPR/Cas9 and demonstrates that the special design of editing sites or the deletion of key gene 1. 3 can reduce MMEJ events and enhance gene editing efficiency. These findings will contribute to advancing CRISPR/Cas-based tools for efficient genome editing in phages and provide a theoretical foundation for the broader application of phages.

Identification and characterization of TatD DNase in planarian Dugesia japonica and its antibiofilm effect.

TatD DNase, a key enzyme in vertebrates and invertebrates, plays a pivotal role in various physiological processes. Dugesia japonica (D. japonica), a flatworm species, has remarkable regenerative capabilities and possesses a simplified immune system. However, the existence and biological functions of TatD DNase in D. japonica require further investigation. Here, we obtained the open reading frame (ORF) of DjTatD and demonstrated its conservation. The three-dimensional structure of DjTatD revealed its active site and binding mechanism. To investigate its enzymological properties, we overexpressed, purified, and characterized recombinant DjTatD (rDjTatD). We observed that DjTatD was primarily expressed in the pharynx and its expression could be significantly challenged upon stimulation with lipopolysaccharide, peptidoglycan, gram-positive and gram-negative bacteria. RNA interference results indicated that both DjTatD and DjDN2s play a role in pharyngeal regeneration and may serve as functional complements to each other. Additionally, we found that rDjTatD and recombinant T7DjTatD effectively reduce biofilm formation regardless of their bacterial origin. Together, our results demonstrated that DjTatD may be involved in the planarian immune response and pharyngeal regeneration. Furthermore, after further optimization in the future, rDjTatD and T7DjTatD can be considered highly effective antibiofilm agents.

Discovery of Two Novel Immunoepitopes and Development of Peptide-based Sarcoidosis Immunoassay.

RATIONALE: Sarcoidosis is a systemic granulomatous disorder associated with hypergammaglobulinemia and the presence of autoantibodies. The specific antigens initiating granulomatous inflammation in sarcoidosis are unknown and there is no specific test available to diagnose sarcoidosis. To discover novel sarcoidosis antigens, we developed a high-throughput T7 phage display library derived from the sarcoidosis cDNA and identified numerous clones differentiating sarcoidosis from other respiratory diseases. After clone sequencing and homology search, we identified two epitopes (Cofilinµ and Chain A) that specifically bind to serum IgGs of sarcoidosis patients. OBJECTIVES: To develop and validate an epitope-specific IgG-based immunoassay specific for sarcoidosis. METHODS: We chemically synthesized both immunoepitopes (Cofilinµ and Chain A), and generated rabbit polyclonal antibodies against both neoantigens. After extensive standardization, we developed a direct peptide ELISA and measured epitope-specific IgG in sera of 386 subjects including, healthy controls (n=100), three sarcoidosis cohorts (n=186), pulmonary tuberculosis (n=70) and lung cancer (n=30). MEASUREMENTS AND MAIN RESULTS: To develop a model to classify sarcoidosis from other groups, data were analyzed using five-fold cross-validation when adjusting for confounders. The Cofilinµ IgGs model yielded a mean sensitivity, specificity, and positive and negative predictive value (PPV, NPV) of 0.97, 0.9, 0.9 and 0.96, respectively. Those same measures for Chain A IgG antibody were 0.9, 0.83, 0.84 and 0.9 respectively. Combining both biomarkers improved AUC, sensitivity, specificity, PPV and NPV. CONCLUSIONS: These results provide a novel immunoassay for sarcoidosis. The discovery of two neoantigens facilitates the development of biospecific drug discovery and the sarcoidosis-specific model.

T7 phage display reveals NOLC1 as a GM3 binding partner in human breast cancer MCF-7 cells.

Ganglioside GM3 is a simple monosialoganglioside (NeuAc-Gal-Glc-ceramide) that modulates cell adhesion, proliferation, and differentiation. Previously, we reported isolation of GM3-binding vascular endothelial growth factor receptor and transforming growth factor-ß receptor by the T7 phage display method (Chung et al., 2009; Kim et al., 2013). To further identify novel proteins interacting with GM3, we extended the T7 phage display method in this study. After T7 phage display biopanning combined with immobilized biotin-labeled 3'-sialyllactose prepared on a streptavidin-coated microplate, we isolated 100 candidate sequences from the human lung cDNA library. The most frequently detected clones from the blast analysis were the human nucleolar and coiled-body phosphoprotein 1 (NOLC1) sequences. We initially identified NOLC1 as a molecule that possibly binds to GM3 and confirmed this binding ability using the glutathione S-transferase fusion protein. Herein, we report another GM3-interacting protein, NOLC1, that can be isolated by the T7 phage display method. These results are expected to be helpful for elucidating the functional roles of ganglioside GM3 with NOLC1. When human breast cancer MCF-7 cells were examined for subcellular localization of NOLC1, immunofluorescence of NOLC1 was observed in the intracellular region. In addition, NOLC1 expression was increased in the nucleolus after treatment with the anticancer drug doxorubicin. GM3 and NOLC1 levels in the doxorubicin-treated MCF-7 cells were correlated, indicating possible associations between GM3 and NOLC1. Therefore, direct interactions between carbohydrates and cellular proteins can pave the path for new signaling phenomena in biology.

Identification of allergenic epitopes destroyed by two processing technologies of glycinin A2 from soybean.

BACKGROUND: Glycinin is one of the most highly allergenic proteins in soybeans, and G2 is one of the five allergenic subunits of glycinin. Compared with the alkaline chain, the acidic chain A2 of the G2 subunit has strong allergenicity. However, the precise epitopes of A2 and the epitopes destroyed during processing are still unknown. RESULTS: In the present study, preparation of two specific antibodies damaged by processing and phage display techniques were applied to locate the antigenic epitopes of glycinin A2 polypeptide chains disrupted by two processing techniques (thermal processing and ultra-high pressure combined thermal processing). Bioinformatics methods were used to predict the possible epitopes of the A2 chain. The A2 chain and its overlapping segments were introduced into T7 phages and expressed on phage shell by phage display. An indirect enzyme-linked immunosorbent assay was used to screen for antigenic epitopes that had been disrupted by the two processing technologies. The results showed that the dominant antigenic region disrupted by processing was located mainly in the A2-3-B fragment. The reacting experiment with the serum of allergic patients showed that the A2-3-B fragment protein was not only an antigenic region, but also an allergenic region. The two processing technologies destroyed the allergenic epitopes of A2 chain, thereby reducing the allergenicity of protein. The amino acids where the dominant allergenic region disrupted by processing was located were: 233 AIVTVKGGLRVTAPAMRKPQQEEDDDDEEEQPQCVE268 . CONCLUSION: Precise epitopes of the acidic chain A2 in glycinin were identified and epitopes destroyed in two common processing methods were also obtained. The application products of rapid detection of de-allergenicity effect of processed food can be developed according to the location of processed destruction allergenic region, which is of great significance with respect to preventing the occurrence of soybean allergenic diseases. © 2022 Society of Chemical Industry.

Recent trends in T7 phage application in diagnosis and treatment of various diseases.

The T7 phage is a virulent phage hosted by Escherichia coli, which poses no threat to animals and plants. Due to the advantages of small genome, well elucidated functional genomics, fast life cycle, and high stability, T7 phage has been widely used in many fields, including biology and medicine. In this review, we focus on the research of T7 phages in biological sciences and medicine, including the application of T7 phages and T7 phage products, T7 phage display systems, and recombinant T7 phages in the treatment and diagnosis of infectious diseases (bacteria, viruses, parasites) and tumor diseases. In addition, we also introduce the therapeutic application of T7 phage in various diseases such as allergic reaction, Alzheimer's disease, inflammatory reaction, and other diseases, and finally discuss the future direction of T7 phage application in the biomedical field.

Protocol for Differential Biopanning of P. falciparum Phage Display cDNA Library to Identify Parasite Targets of Protective Antibodies.

Malaria remains a significant global health burden, killing hundreds of thousands of children annually (WHO, The world malaria report. WHO, Geneva, 2019). Despite decades of effort, no broadly effective vaccine exists. Differential screening of parasite phage display libraries is a promising approach to identify the targets of human antibodies expressed by resistant but not by susceptible individuals (Raj et al., Nature, 582, 104-108, 2020; Science, 344, 871-877, 2014). Our whole proteome differential screening (WPDS) approach consists of positive selection to capture phage that bind antibodies expressed by malaria-resistant individuals, followed by negative selection to remove phage that bind antibodies expressed by malaria-susceptible individuals, and amplification of differentially recognized clones.

Rapid 40 kb Genome Construction from 52 Parts through Data-optimized Assembly Design.

Large DNA constructs (>10 kb) are invaluable tools for genetic engineering and the development of therapeutics. However, the manufacture of these constructs is laborious, often involving multiple hierarchical rounds of preparation. To address this problem, we sought to test whether Golden Gate assembly (GGA), an in vitro DNA assembly methodology, can be utilized to construct a large DNA target from many tractable pieces in a single reaction. While GGA is routinely used to generate constructs from 5 to 10 DNA parts in one step, we found that optimization permitted the assembly of >50 DNA fragments in a single round. We applied these insights to genome construction, successfully assembling the 40 kb T7 bacteriophage genome from up to 52 parts and recovering infectious phage particles after cellular transformation. The assembly protocols and design principles described here can be applied to rapidly engineer a wide variety of large and complex assembly targets.

Autoantibodies against cytoskeletons and lysosomal trafficking discriminate sarcoidosis from healthy controls, tuberculosis and lung cancers.

Sarcoidosis is a systemic granulomatous disease of unknown etiology. Hypergammaglobulinemia and the presence of autoantibodies in sarcoidosis suggest active humoral immunity to unknown antigen(s). We developed a complex cDNA library derived from tissues of sarcoidosis patients. Using a high throughput method, we constructed a microarray platform from this cDNA library containing large numbers of sarcoidosis clones. After selective biopanning, 1070 sarcoidosis-specifc clones were arrayed and immunoscreend with 152 sera from patients with sarcoidosis and other pulmonary diseases. To identify the sarcoidosis classifiers two statistical approaches were conducted: First, we identified significant biomarkers between sarcoidosis and healthy controls, and second identified markers comparing sarcoidosis to all other groups. At the threshold of an False Discovery Rate (FDR) < 0.01, we identified 14 clones in the first approach and 12 clones in the second approach discriminating sarcoidosis from other groups. We used the classifiers to build a naïve Bayes model on the training-set and validated it on an independent test-set. The first approach yielded an AUC of 0.947 using 14 significant clones with a sensitivity of 0.93 and specificity of 0.88, whereas the AUC of the second option was 0.92 with a sensitivity of 0.96 and specificity of 0.83. These results suggest robust classifier performance. Furthermore, we characterized the informative phage clones by sequencing and homology searches. Large numbers of classifier-clones were peptides involved in cellular trafficking and cytoskeletons. These results show that sarcoidosis is associated with a specific pattern of immunoreactivity that can discriminate it from other diseases.

RNA Management During T7 Infection.

Post-transcriptional regulation (PTR) determines the fate of RNA in the cell and represents an important control point in the flow of genetic information and thus underpins many, if not all, aspects of cell function. Host takeover by phages through misappropriation of the bacterial transcription machinery is a relatively advanced area of research. However, several phages encode small regulatory RNAs, which are major mediators of PTR, and produce specific proteins to manipulate bacterial enzymes involved in RNA degradation.1-4 However, PTR during phage development still represents an understudied area of phage-bacteria interaction biology. In this study, we discuss the potential role PTR could play in determining the fate of RNA during the lifecycle of the prototypic phage T7 in Escherichia coli.

T7 Phage as an Emerging Nanobiomaterial with Genetically Tunable Target Specificity.

Bacteriophages, also known as phages, are specific antagonists against bacteria. T7 phage has drawn massive attention in precision medicine owing to its distinctive advantages, such as short replication cycle, ease in displaying peptides and proteins, high stability and cloning efficiency, facile manipulation, and convenient storage. By introducing foreign gene into phage DNA, T7 phage can present foreign peptides or proteins site-specifically on its capsid, enabling it to become a nanoparticle that can be genetically engineered to screen and display a peptide or protein capable of recognizing a specific target with high affinity. This review critically introduces the biomedical use of T7 phage, ranging from the detection of serological biomarkers and bacterial pathogens, recognition of cells or tissues with high affinity, design of gene vectors or vaccines, to targeted therapy of different challenging diseases (e.g., bacterial infection, cancer, neurodegenerative disease, inflammatory disease, and foot-mouth disease). It also discusses perspectives and challenges in exploring T7 phage, including the understanding of its interactions with human body, assembly into scaffolds for tissue regeneration, integration with genome editing, and theranostic use in clinics. As a genetically modifiable biological nanoparticle, T7 phage holds promise as biomedical imaging probes, therapeutic agents, drug and gene carriers, and detection tools.

Evaluation of dendritic cell-targeting T7 phages as a vehicle to deliver avian influenza virus H5 DNA vaccine in SPF chickens.

Introduction: There is a growing demand for effective technologies for the delivery of antigen to antigen-presenting cells (APCs) and their immune-activation for the success of DNA vaccines. Therefore, dendritic cell (DC)-targeting T7 phages were used as a vehicle to deliver DNA vaccine. Methods: In this study, a eukaryotic expression plasmid pEGFP-C1-HA2-AS containing the HA2 gene derived from the avian H5N1 virus and an anchor sequence (AS) gene required for the T7 phage packaging process was developed. To verify the feasibility of phage delivery, the plasmid encapsulated in DC-targeting phage capsid through the recognition of AS was evaluated both in vitro and in vivo. The pEGFP-C1-HA2-AS plasmid could evade digestion by DNase I by becoming encapsulated into the phage particles and efficiently expressed the HA2 antigen in DCs with the benefit of DC-targeting phages. Results: For chickens immunized with the DC-targeting phage 74 delivered DNA vaccine, the levels of IgY and IgA antibodies, the concentration of IFN-γ and IL-12 cytokines in serum, the proliferation of lymphocytes, and the percentage of CD4+/CD8+ T lymphocytes isolated from peripheral blood were significantly higher than chickens which were immunized with DNA vaccine that was delivered by non-DC-targeting phage or placebo (p<0.05). Phage 74 delivered one-fiftieth the amount of pEGFP-C1-HA2-AS plasmid compared to Lipofectin, however, a comparable humoral and cellular immune response was achieved. Although, the HA2 DNA vaccine delivered by the DC-targeting phage induced enhanced immune responses, the protection rate of virus challenge was not evaluated. Conclusion: This study provides a strategy for development of a novel avian influenza DNA vaccine and demonstrates the potential of DC-targeting phage as a DNA vaccine delivery vehicle.

Mycoplasma genitalium Protein of Adhesion Promotes the Early Proliferation of Human Urothelial Cells by Interacting with RPL35.

Mycoplasma genitalium is a newly recognized pathogen associated with sexually transmitted diseases (STDs). MgPa, the adhesion protein of Mycoplasma genitalium, is the main adhesin and the key factor for M. genitalium interacting with host cells. Currently, the long-term survival mechanism of M. genitalium in the host is not clear. In this study, a T7 phage-displayed human urothelial cell (SV-HUC-1) cDNA library was constructed, and the interaction of MgPa was screened from this library using the recombinant MgPa (rMgPa) as a target molecule. We verified that 60S ribosomal protein L35 (RPL35) can interact with MgPa using far-Western blot and co-localization analysis. According to the results of tandem mass tag (TMT) labeling and proteome quantitative analysis, there were altogether 407 differentially expressed proteins between the pcDNA3.1(+)/MgPa-transfected cells and non-transfected cells, of which there were 6 downregulated proteins and 401 upregulated proteins. The results of qRT-PCR demonstrated that interaction between rMgPa and RPL35 could promote the expressions of EIF2, SRP68, SERBP1, RPL35A, EGF, and TGF-ß. 3-(4,5)-Dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide bromide (MTT) assays corroborated that the interaction between rMgPa and RPL35 could promote SV-HUC-1 cell proliferation. Therefore, our findings indicated that the interaction between rMgPa and RPL35 can enhance the expressions of transcription-initiation and translation-related proteins and thus promote cell proliferation. This study elucidates a new biological function of MgPa and can explain this new mechanism of M. genitalium in the host.

Biological Characterization and Evolution of Bacteriophage T7-△holin During the Serial Passage Process.

Bacteriophage T7 gene 17.5 coding for the only known holin is one of the components of its lysis system, but the holin activity in T7 is more complex than a single gene, and evidence points to the existence of additional T7 genes with holin activity. In this study, a T7 phage with a gene 17.5 deletion (T7-△holin) was rescued and its biological characteristics and effect on cell lysis were determined. Furthermore, the genomic evolution of mutant phage T7-△holin during serial passage was assessed by whole-genome sequencing analysis. It was observed that deletion of gene 17.5 from phage T7 delays lysis time and enlarges the phage burst size; however, this biological characteristic recovered to normal lysis levels during serial passage. Scanning electron microscopy showed that the two opposite ends of E. coli BL21 cells swell post-T7-△holin infection rather than drilling holes on cell membrane when compared with T7 wild-type infection. No visible progeny phage particle accumulation was observed inside the E. coli BL21 cells by transmission electron microscopy. Following serial passage of T7-△holin from the 1st to 20th generations, the mRNA levels of gene 3.5 and gene 19.5 were upregulated and several mutation sites were discovered, especially two missense mutations in gene 19.5, which indicate a potential contribution to the evolution of the T7-△holin. Although the burst size of T7-△holin increased, high titer cultivation of T7-△holin was not achieved by optimizing the culture process. Accordingly, these results suggest that gene 19.5 is a potential lysis-related component that needs to be studied further and that the T7-△holin strain with its gene 17.5 deletion is not adequate to establish the high-titer phage cultivation process.

Homing Peptides for Cancer Therapy.

Tumor-homing peptides are widely used for improving tumor selectivity of anticancer drugs and imaging agents. The goal is to increase tumor uptake and reduce accumulation at nontarget sites. Here, we describe current approaches for tumor-homing peptide identification and validation, and provide comprehensive overview of classes of tumor-homing peptides undergoing preclinical and clinical development. We focus on unique mechanistic features and applications of a recently discovered class of tumor-homing peptides, tumor-penetrating C-end Rule (CendR) peptides, that can be used for tissue penetrative targeting of extravascular tumor tissue. Finally, we discuss unanswered questions and future directions in the field of development of peptide-guided smart drugs and imaging agents.

Production, characterization and in-vitro applications of single-domain antibody against thyroglobulin selected from novel T7 phage display library.

Single- domain antibodies (SdAbs) have been deployed in various biomedical applications in the recent past. However, there are no reports of their use in the immunoradiometric assays (IRMA) for thyroglobulin (Tg). Tg is the precursor molecule for the biosynthesis of thyroid hormones: thyroxine and triiodothyronine, which are essential for the regulation of normal metabolism in all vertebrates. Patients with differentiated thyroid cancer (DTC) require periodic monitoring of their serum thyroglobulin levels, as it serves as a prognostic marker for DTC. Here, we report a methodology to produce SdAbs against human-Tg, by a hybrid immunization/directed-evolution approach by displaying the SdAb gene-repertoire derived from a hyperimmune camel in the T7 phage display system. We have demonstrated the immunoreactivity of anti-Tg-SdAb (KT75) in immunoassays for thyroglobulin and measured its affinity by surface plasmon resonance (KD ~ 18 picomolar). Additionally, we have shown the quantitative-binding property of SdAb for the first time in IRMA for thyroglobulin. The serum Tg values obtained from SdAb-Tg-IRMA and in-house assay using murine anti-Tg-monoclonal antibody as tracer significantly correlated, r = 0.81, p < 0.05. Our results highlight the scope of using the T7 phage display system as an alternative for the conventional M13-phage to construct single-domain antibody display libraries.

Combining the advantages of prokaryotic expression and T7 phage display systems to obtain antigens for antibody preparation.

The gene encoding the phage major capsid protein 10A was cloned into the prokaryotic expression vector pET24a, and a 6XHis-tag was fused to the 3'-end of the 10A gene to verify complete expression. The recombinant plasmid was transformed into Escherichia coli (E. coli) BL21 (DE3) cells, and 10A expression was induced by IPTG. SDS-PAGE and Western blot were used to confirm the target protein expression. The T7Select10-3b vector was added to the cultured bacteria expressing 10A at a multiplicity of infection (MOI) ranging from 0.01 to 0.1, and complete lysis of the bacteria was monitored by absorbance changes in the medium. The recombinant phage (reP) was harvested by PEG/NaCl sedimentation and resuspended in PBS. ELISA was performed to verify the presence of the 6XHis-tag on the surface of reP. The 10A-fusion expression vectors (pET10A-flag, pET10A-egfp, and pET10A-pct) were constructed, and fusion proteins were expressed and detected by the same method. The corresponding rePs (reP-Flag, reP-EGFP, and reP-PCT) were prepared by T7Select10-3b infection. After the expression of the peptides/proteins on the reP surfaces was confirmed, reP-Flag and reP-PCT were used to immunize mice to prepare anti-Flag and anti-PCT antibodies. The results showed that rePs prepared using the 10A-fusion vector and T7Select10-3b can be used as antigens to immunize mice and prepare antibodies. This method may be able to meet the rapid antigen preparation requirements for antibody production. Notably, the recombinant phage (reP) described in this study was obtained by the sedimentation method from T7Select10-3b-infected E. coli BL21 (DE3) cells carrying the major capsid protein 10A expression vector or 10A-fusion protein vector.

The RNA-binding protein Hfq assembles into foci-like structures in nitrogen starved Escherichia coli.

The initial adaptive responses to nutrient depletion in bacteria often occur at the level of gene expression. Hfq is an RNA-binding protein present in diverse bacterial lineages that contributes to many different aspects of RNA metabolism during gene expression. Using photoactivated localization microscopy and single-molecule tracking, we demonstrate that Hfq forms a distinct and reversible focus-like structure in Escherichia coli specifically experiencing long-term nitrogen starvation. Using the ability of T7 phage to replicate in nitrogen-starved bacteria as a biological probe of E. coli cell function during nitrogen starvation, we demonstrate that Hfq foci have a role in the adaptive response of E. coli to long-term nitrogen starvation. We further show that Hfq foci formation does not depend on gene expression once nitrogen starvation has set in and occurs indepen-dently of the transcription factor N-regulatory protein C, which activates the initial adaptive response to N starvation in E. coli These results serve as a paradigm to demonstrate that bacterial adaptation to long-term nutrient starvation can be spatiotemporally coordinated and can occur independently of de novo gene expression during starvation.

Bacteriophage Inspired Growth-Decoupled Recombinant Protein Production in Escherichia coli.

Modulating resource allocation in bacteria to redirect metabolic building blocks to the formation of recombinant proteins rather than biomass formation remains a grand challenge in biotechnology. Here, we present a novel approach for improved recombinant protein production (RPP) using Escherichia coli (E. coli) by decoupling recombinant protein synthesis from cell growth. We show that cell division and host mRNA transcription can be successfully inhibited by coexpression of a bacteriophage-derived E. coli RNA polymerase (RNAP) inhibitor peptide and that genes overtranscribed by the orthogonal T7 RNAP can finally account to >55% of cell dry mass (CDM). This RNAP inhibitor peptide binds the E. coli RNAP and therefore prevents σ-factor 70 mediated formation of transcriptional qualified open promoter complexes. Thereby, the transcription of σ-factor 70 driven host genes is inhibited, and metabolic resources can be exclusively utilized for synthesis of the protein of interest (POI). Here, we mimic the late phase of bacteriophage infection by coexpressing a phage-derived xenogeneic regulator that reprograms the host cell and thereby are able to significantly improve RPP under industrial relevant fed-batch process conditions at bioreactor scale. We have evaluated production of several different recombinant proteins at different scales (from microscale to 20 L fed-batch scale) and have been able to improve total and soluble proteins yields up to 3.4-fold in comparison to the reference expression system E. coli BL21(DE3). This novel approach for growth-decoupled RPP has profound implications for biotechnology and bioengineering and helps to establish more cost-effective and generic manufacturing processes for biologics and biomaterials.

Identification of antigenic sites destructed by high hydrostatic pressure (HHP) of the ß subunit of ß-conglycinin.

ß-conglycinin is one of the most allergenic proteins, and its constituent subunits α', α, and ß are all potential allergens to humans. In the present study, we concentrated on the destructed antigenic sites of ß subunit of ß-conglycinin after high hydrostatic pressure (HHP) treatment. In this paper, the overlapping gene fragments of the ß subunit of ß-conglycinin were amplified by polymerase chain reaction (PCR) and cloned into T7 phage vectors. After being packaged in vitro, the recombinant T7 phage was constructed, and the overlapping fragments of the ß subunit were displayed on the phage surface. The recombinant phages that expressed the overlapping fragments of the ß subunit were used to react with specific antiserum by indirect ELISA to identify the HHP destructed antigenic sites. After three rounds of expression and identification, we used synthetic peptide technology to identify that the obtained fragment was a conformational epitope. We further confirmed that HHP treatment changed the conformational structure of ß-conglycinin, which reduced the antigenicity of the protein.