Co-expression of recombinant single chain variable fragment recognizing blood antigen fused with sumo and chaperones in Escherichia coli

2018 ◽  
Vol 40 (4) ◽  
Author(s):  
Dang Thi Ngoc Ha ◽  
Le Thi Thu Hong ◽  
Truong Nam Hai
Keyword(s):  
Recombinant Antibody ◽  
Blood Type ◽  
Soluble Form ◽  
Supernatant Fraction ◽  
Blood Group Antigens ◽  
Single Chain ◽  
E Coli ◽  
Recombinant Scfv ◽  
Single Chain Variable Fragments

Single chain variable fragments (scFv) have widely been used in research, diagnosis and treatment, but the scFv is considered as difficult protein for expression in E. coli. In previous studies, we expressed a construction of recombinant single chain variable fragments again antigen specific for blood type A (antiA-scFv) individually or fused with Trx or SUMO. However, soluble fraction was low abandant and only approximately 40% when fused with Trx, the other cases were expressed in form of inclusion body. Therefore, it was difficult for purification, refolding and activity assesment. In thispaper, we demonstrated a suitable construction for soluble production of antiA-scFv fused with SUMO (SM/antiA-scFv) in presence of chaparones. Under fermentation with 0.1 mM IPTG at 20oC, the SM/antiA-scFv was entirely expressed in soluble form. Importantly, after cleavage from SUMO with SUMOprotease, antiA-scFv was still maintained in the supernatant fraction. Therefore, it can help ensure bioactivity and is useful for purification process. To the best of our knowledge, this is the first report showing soluble recombinant scFv fused with SUMO in presence of chaperone for determination of blood group antigens. Thus, this result facilitates the optimal study of soluble expression, purification and bioactivity determination of the antiA-scFv recombinant antibody. 

scholarly journals Expression of the recombinant single chain variable fragments recognizing blood antigen fused with thioredoxin in Escherichia coli

2019 ◽  
Vol 41 (1) ◽  
Author(s):  
Dang Thi Ngoc Ha ◽  
Le Thi Thu Hong ◽  
Truong Nam Hai
Keyword(s):  
Expression Vector ◽  
Recombinant Antibody ◽  
Soluble Form ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
E Coli ◽  
Recombinant Scfv ◽  
Insoluble Form ◽  
Single Chain Variable Fragments

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. 

scholarly journals High-yield expression of periplasmic single-chain variable fragments by solid Escherichia coli cultures

BioTechniques ◽  
2021 ◽  
Author(s):  
Yoshiro Hanyu ◽  
Mieko Kato
Keyword(s):  
Escherichia Coli ◽  
Liquid Culture ◽  
Recombinant Antibody ◽  
Antibody Fragments ◽  
High Yield ◽  
Single Chain ◽  
E Coli ◽  
Recombinant Antibody Fragments ◽  
Single Chain Variable Fragments ◽  
High Yields

High-yield expression of quality antibody fragments is indispensable for research and diagnosis. Most recombinant antibody fragments are expressed in Escherichia coli using liquid cultures; however, their yields and quality are often poor. Here the authors expressed a single-chain variable fragment in E. coli cultivated on the wet surface of a solid support. Compared with a liquid culture, the authors obtained 2.5-times more single-chain variable fragments with membrane-cultivated E. coli. This method has two important advantages: it enables high yields of periplasmic single-chain variable fragments compared with liquid culture and offers simple and rapid expression and extraction.

scholarly journals Production of a Soluble Recombinant Antibody Fragment against MMP9 Using Escherichia coli

Medicina ◽  
2021 ◽  
Vol 57 (9) ◽  
pp. 981
Author(s):  
Chang-Hun Yeom ◽  
Hee-Jin Jeong
Keyword(s):  
Escherichia Coli ◽  
Recombinant Antibody ◽  
Antibody Fragment ◽  
Soluble Form ◽  
Efficient Production ◽  
Binding Ability ◽  
Recombinant Scfv ◽  
Recombinant Antibody Fragment ◽  
Recombinant Antibody Fragments ◽  
Metalloproteinase 9

Matrix metalloproteinase 9 (MMP9) is involved in several aspects of the pathology of cancer, including invasion, metastasis, and angiogenesis. In this study, we expressed a recombinant scFv-type anti-MMP9 antibody in soluble form using Escherichia coli, purified it, and confirmed its antigen-binding ability. The convenient, rapid, inexpressive system used in this study for producing recombinant antibody fragments needs only five days, and thus can be used for the efficient production of scFv against MMP9, which can be used in a range of applications and industrial fields, including diagnosis and treatment of inflammatory and cancer-related diseases.

scholarly journals Expression of Overlapping PreS1 Fragment Recombinant Proteins for the Determination of Immunogenic Domains in HBsAg PreS1 Region

2004 ◽  
Vol 36 (6) ◽  
pp. 397-404 ◽  
Author(s):  
Wei-Guo Hu ◽  
Jun Wei ◽  
Xin-Xiu Yang ◽  
Heng-Chuan Xia ◽  
Feng Li ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Fusion Proteins ◽  
Competitive Elisa ◽  
Soluble Form ◽  
Immunological Property ◽  
E Coli ◽  
C Terminus ◽  
New Vaccines ◽  
Gst Fusion Proteins

Abstract In this study, eight preS1 fragments overlapped in preS1 (21–119) region of HBV adr subtype, i.e. preS1 (21–47), preS1 (34–59), preS1 (48–70), preS1 (60–85), preS1 (71–94), preS1 (86–109), preS1 (95–119) and preS1 (21–119), were cloned by PCR, and expressed as GST fusion proteins. These GST-preS1 fusion proteins were highly expressed in soluble form in E. coli, and about 50 to 90 mg soluble fusion proteins were purified from 1 L culture. Using these fusion proteins, the immunogenic domains in preS1 (21–119) region were identified by Western blot analysis and competitive ELISA. The results showed that the immunogenic domains mainly existed in preS1 (21–59) in N-terminus and preS1 (95–109) in C-terminus, and more importantly, a major immunogenic domain preS1 (34–59), which has much stronger immunogenicity, was identified. It was also supported by the predictions of secondary structure and immunological property in the preS1 (21–119) region. The results here would be helpful for the design of new vaccines against HBV.

scholarly journals Single Chain Variable Fragments Produced in Escherichia coli against Heat-Labile and Heat-Stable Toxins from Enterotoxigenic E. coli

PLoS ONE ◽  
2015 ◽  
Vol 10 (7) ◽  
pp. e0131484 ◽  
Author(s):  
Christiane Y. Ozaki ◽  
Caio R. F. Silveira ◽  
Fernanda B. Andrade ◽  
Roberto Nepomuceno ◽  
Anderson Silva ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Single Chain ◽  
E Coli ◽  
Heat Stable ◽  
Heat Labile ◽  
Single Chain Variable Fragments

scholarly journals Purification and Characterization of Antibodies in Single-Chain Format against the E6 Oncoprotein of Human Papillomavirus Type 16

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
F. Verachi ◽  
Z. Percario ◽  
P. Di Bonito ◽  
E. Affabris ◽  
C. Amici ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Cancer Cells ◽  
Antibody Fragment ◽  
Human Papillomaviruses ◽  
Soluble Form ◽  
Single Chain ◽  
Binding Studies ◽  
E Coli ◽  
E6 Oncoprotein ◽  
Cervical Cancer Cells

In Human Papillomaviruses- (HPV-) associated carcinogenesis, continuous expression of the E6 oncoprotein supports its value as a potential target for the development of diagnostics and therapeutics for HPV cancer. We previously reported that the I7 single-chain antibody fragment (scFv) specific for HPV16 E6, expressed as an intrabody by retroviral system, could inhibit significantly the growth of cervical cancer cells in vitro and was even able to reduce tumor development in experimental HPV-related cancer models. Nevertheless, for the development of therapeutic tools to be employed in humans, it is important to achieve maximum safety guarantee, which can be provided by the protein format. In the current study, two anti-16E6 scFvs derived from I7 were expressed in E. coli and purified in soluble form by affinity chromatography. Specificity, sensitivity and stability in physiologic environment of the purified scFvs were demonstrated by binding studies using recombinant 16E6 as an antigen. The scFvs functionality was confirmed by immunofluorescence in cervical cancer cells, where the scFvs were able to recognize the nuclear E6. Furthermore, an antiproliferative activity of the scFvI7nuc delivered in protein format to HPV16-positive cell lines was observed. Our results demonstrate that functional anti-16E6 scFvs can be produced in E. coli, suggesting that such purified antibodies could be used in the diagnosis and treatment of HPV-induced malignancies.

scholarly journals Generation and functional characterization of a single-chain variable fragment (scFv) of the anti-FGF2 3F12E7 monoclonal antibody

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Rodrigo Barbosa de Aguiar ◽  
Tábata de Almeida da Silva ◽  
Bruno Andrade Costa ◽  
Marcelo Ferreira Marcondes Machado ◽  
Renata Yoshiko Yamada ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Functional Characterization ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Variable Regions ◽  
Recombinant Scfv ◽  
Antitumor Potential ◽  
Single Chain Variable Fragments ◽  
Binding Determinants

AbstractSingle-chain variable fragments (scFvs) are small-sized artificial constructs composed of the immunoglobulin heavy and light chain variable regions connected by a peptide linker. We have previously described an anti-fibroblast growth factor 2 (FGF2) immunoglobulin G (IgG) monoclonal antibody (mAb), named 3F12E7, with notable antitumor potential revealed by preclinical assays. FGF2 is a known angiogenesis-associated molecule implicated in tumor progression. In this report, we describe a recombinant scFv format for the 3F12E7 mAb. The results demonstrate that the generated 3F12E7 scFv, although prone to aggregation, comprises an active anti-FGF2 product that contains monomers and small oligomers. Functionally, the 3F12E7 scFv preparations specifically recognize FGF2 and inhibit tumor growth similar to the corresponding full-length IgG counterpart in an experimental model. In silico molecular analysis provided insights into the aggregation propensity and the antigen-recognition by scFv units. Antigen-binding determinants were predicted outside the most aggregation-prone hotspots. Overall, our experimental and prediction dataset describes an scFv scaffold for the 3F12E7 mAb and also provides insights to further engineer non-aggregated anti-FGF2 scFv-based tools for therapeutic and research purposes.

Molecular Cloning and Expression of Recombinant Phage Antibody against Fumonisin B1

1996 ◽  
Vol 59 (11) ◽  
pp. 1208-1212 ◽  
Author(s):  
HUI-REN ZHOU ◽  
JAMES J. PESTKA ◽  
L. PATRICK HART
Keyword(s):  
Polymerase Chain Reaction Amplification ◽  
Recombinant Antibody ◽  
Fumonisin B1 ◽  
Cloning And Expression ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Recombinant Phage ◽  
Recombinant Scfv ◽  
Food Borne ◽  
Variable Heavy

Bacteriophage expression systems offer promise for the development of novel antibody reagents applicable to detection of microbial agents and their toxins in foods. In this study, fumonisin B1 (FB1)-specific antibodies, composed of a single chain containing a variable heavy (VH) and light (VL) chain fragments (ScFv), were cloned using mRNA from either spleen cells of mice immunized with FB1-BSA conjugate or from an existing hybridoma cell line that produces anti-FB1 antibody. The approach consisted of (i) reverse transcription of isolated mRNA, (ii) polymerase chain reaction amplification of VH and VL cDNAs, (iii) ligation of VL and VH chains, and (iv) expression of ScFv proteins on the surface of a filamentous bacteriophage or as a soluble fragment. The efficacy of using the recombinant ScFv proteins for the detection of FB1 were evaluated in a competitive indirect enzyme-linked immunosorbent assay (CI-ELISA). Compared to polyclonal or monoclonal antibodies for FB1, the recombinant ScFv proteins were 10 to 100 times less sensitive in the CI-ELISA. The secreted ScFv protein did not cross-react with FB2 or FB3. The results indicated that production of a recombinant antibody to a mycotoxin is feasible. However, problems associated with the affinity or avidity of ScFv fragments need to be further addressed before this technology is adaptable to the development of improved immunodiagnostic kits for mycotoxins or other food borne disease hazards.

Preparation of single-chain Fv antibodies in the cytoplasm of Escherichia coli by simplified and systematic chaperone optimization

2019 ◽  
Vol 166 (6) ◽  
pp. 455-462 ◽  
Author(s):  
Chenjiang Liu ◽  
Yoshihiro Kobashigawa ◽  
Soichiro Yamauchi ◽  
Yuya Toyota ◽  
Manaka Teramoto ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Tertiary Structure ◽  
Guanidine Hydrochloride ◽  
Molecular Size ◽  
Soluble Form ◽  
Single Chain ◽  
Variable Regions ◽  
E Coli ◽  
Disulphide Bonds ◽  
Single Chain Fv

Abstract A single-chain variable fragment (scFv) antibody is a recombinant protein in which a peptide linker connects the variable regions of the heavy chain and light chain. Due to its smaller molecular size, an scFv can be expressed using Escherichia coli. The presence of two disulphide bonds in the molecule often prevents expression of correctly folded scFv in the E. coli cytoplasm, making a refolding process necessary to regenerate scFv activity. The refolding process is time-consuming and requires large amounts of expensive reagents, such as guanidine hydrochloride, l-arginine and glutathione. Here, to conveniently obtain scFv proteins, we devised a simple and systematic method to optimize the co-expression of chaperone proteins and to combine them with specially engineered E. coli strains that permit the formation of stable disulphide bonds within the cytoplasm. Several scFv proteins were successfully obtained in a soluble form from E. coli cytoplasm. Thermal denaturation experiments and/or surface plasmon resonance measurements revealed that the thus-obtained scFvs possessed a stable tertiary structure and antigen-binding activity. The combined use of engineered E. coli with the simplified and systematic chaperone optimization can be useful for the production of scFv proteins.

scholarly journals Generation of recombinant scFv antibody against Ochratoxin A (OTA)

2018 ◽  
Vol 22 (2) ◽  
pp. 107 ◽  
Author(s):  
Ranya Pranomphon ◽  
Witsanu Srila ◽  
Montarop Yamabhai
Keyword(s):  
Ochratoxin A ◽  
Recombinant Antibody ◽  
Cross Reactivity ◽  
Agricultural Product ◽  
Binding Property ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Recombinant Scfv ◽  
Contaminated Food ◽  
Soluble Scfv

Ochratoxin A (OTA) is a mycotoxin commonly found in agricultural products and can accumulate in the blood and tissues after that consuming contaminated food. Recombinant single-chain antibody fragments (scFv) against OTA were selected from phage display libraries. After of one round of biopanning against BSA-conjugated OTA (OTA-BSA), 52 and 6 phage clones displaying scFv antibodies were isolated from human (Yamo I.3) and rabbit (Bozmix I.2) libraries. Two phage clones (one from each libraries, i.e., yOTA1e3 and bOTA2a9) showed binding to free toxin by competitive ELISA. The soluble scFv antibodies were produced by superinfecting phage clones into E. coli suppressor strain HB2151. The scFv genes from these two phage clones were sub-cloned into pKP300ΔIII vectors to generate scFv-AP fusions. The binding affinity (IC50) of antibody derived from human library was higher than those from rabbit library. The binding property of recombinant antibody in the form of scFv-AP was better than those of soluble scFv form. Cross-reactivity analysis indicated that the two recombinant antibodies did not cross-react with other soluble toxins, namely AFB1, DON, ZEN and FB. The ability to use the recombinant scFv-AP to detect contaminated toxins in agricultural product (corn) was demonstrated.

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