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

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.

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

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.

NGHIÊN CỨU TỐI ƯU ĐIỀU KIỆN BIỂU HIỆN INTERLEUKIN-3 NGƯỜI DUNG HỢP VỚI PelB TRONG E. coli

Interleukin-3 (IL-3) is a multifunctional cytokine which modulates the proliferation, differentiation and maturation of various types of hematopoietic cells. Gene coding for IL-3 linked with pelB signal were incorporated into pET22b(+) for  expression of il3 gene in E. coli BL21; but IL-3 was synthesized at very low levels and still in fusion with PelB. To facilitate purification process, finding the appropriate fermentation conditions plays a key role in order to enhance gene il-3 expression and cleavage of PelB. In this study, we have optimized the conditions for the expression of IL-3, which included E. coli host strain JM109, LB cultivation medium, induction temperature was 25oC; induction with 0.05 mM IPTG at OD600 = 1. The cell biomass increases at about 2.3 times after optimization. IL-3 protein was expressed in the form of inclusion body and the PelB signal was cleaved. This result is conducive for purification of large amount of IL-3 to determine characteristics of protein. Interleukin-3 người (IL-3) là một cytokine đa chức năng tham gia vào các quá trình tự đổi mới, nhân lên, biệt hóa và trưởng thành của nhiều loại tế bào máu. Sau khi đưa gen il-3 gắn thêm tín hiệu tiết pelB vào vector pET22b(+) và tiến hành biểu hiện ở chủng E. coli BL21, chúng tôi nhận thấy IL-3 được tổng hợp ở mức rất thấp và còn gắn với PelB. Để thuận tiện cho khâu tinh sạch thì vấn đề then chốt là nghiên cứu tìm ra các điều kiện phù hợp làm tăng lượng IL-3 được tổng hợp, đồng thời cắt được PelB. Trong nghiên cứu này, chúng tôi so sánh khả năng sinh tổng hợp IL-3 của các chủng E. coli BL21, JM109, Soluble và Rossetta2; sau đó tối ưu hóa điều kiện biểu hiện gen il-3 về thành phần môi trường, nhiệt độ, nồng độ IPTG, thời điểm cảm ứng và kiểm tra trạng thái tồn tại của IL-3. Kết quả thu được cho thấy gen il-3 biểu hiện tốt và ổn định nhất ở chủng E. coli JM109. Dưới các điều kiện lên men thích hợp trong môi trường LB, ở 25oC, cảm ứng 0,05 mM IPTG tại OD600=1, IL-3 biểu hiện tốt, cắt khỏi PelB và tồn tại ở trạng thái không tan trong tế bào chất. Sinh khối tế bào tăng lên khoảng 2,3 lần sau khi tối ưu. Kết quả này là tiền đề cho bước tinh sạch lượng lớn IL-3 cho nghiên cứu tính chất của protein

Hydrogen Gas Sensing Using Palladium-Graphene Nanocomposite Material Based on Surface Acoustic Wave

We report the fabrication and characterization of surface acoustic wave (SAW) hydrogen sensors using palladium-graphene (Pd-Gr) nanocomposite as sensing material. The Pd-Gr nanocomposite as sensing layer was deposited onto SAW delay line sensor-based interdigitated electrodes (IDTs)/aluminum nitride (AlN)/silicon (Si) structure. The Pd-Gr nanocomposite was synthesized by a chemical route and deposited onto SAW sensors by air-brush spraying. The SAW H2 sensor using Pd-Gr nanocomposite as a sensing layer shows a frequency shift of 25 kHz in 0.5% H2 concentration at room temperature with good repeatability and stability. Moreover, the sensor showed good linearity and fast response/recovery within ten seconds with various H2 concentrations from 0.25 to 1%. The specific interaction between graphene and SAW transfer inside AlN/Si structures yields a high sensitivity and fast response/recovery of SAW H2 sensor based on Pd-Gr/AlN/Si structure

Magnetic Properties of FePt Nanoparticles Prepared by Sonoelectrodeposition

Sonoelectrodeposition is a useful technique to make metallic nanoparticles, using ultrasound during electrodeposition to remove nanoparticles as they grow on the cathode surface. This paper reports some structural and magnetic properties of FePt nanoparticles prepared by this method. The as-prepared Fe45Pt55 nanoparticles were ferromagnetic at room temperature. Upon annealing at 700°C for 1 h under H2 atmosphere, the saturation magnetization and the coercivity of the nanoparticles were improved significantly. The annealed nanoparticles showed a high coercivity of 13.5 kOe at 2 K and of 9 kOe at room temperature. Sonoelectrodeposition is a promising technique to make large quantity of FePt nanoparticles

A comparison between Hydrochloric acid and Trifluoroacetic acid in hydrolysis method of exopolysaccharide from Ophiocordyceps sinensis in Monosaccharide composition analysis by GC-FID

The monosaccharide composition is one of the crucial factors affecting the bioactivity of exopolysaccharide (EPS) in Cordyceps species. Therefore, many scientists have studied, analyzed monosaccharide composition and structure of EPS from Cordyceps species, especially Ophiocordyceps sinensis (O. sinensis). This study aimed to compare hydrochloric acid (HCl) with trifluoroacetic acid (TFA) in the EPS hydrolysis process in monosaccharide composition analysis by Gas Chromatography with Flame-Ionization Detection (GC-FID). The hydrolysis is a crucial step in forming the acetyl derivative, which helps the GC-FID technique to have good results in monosaccharide composition analysis. The results showed that hydrolysis with HCl gave a higher hydrolysis efficiency and was more suitable than hydrolysis by TFA in pretreatment to EPS for GC-FID. Hydrolysis results were analyzed through thin-layer chromatography and high-performance liquid chromatography (HPLC), then Acetyl derivatives were produced and finally analyzed by GC-FID to determine the monosaccharide composition of EPS. For EPS hydrolyzed by HCl, the analytical results presented that this sample had 6 kinds of monosaccharides, including rhamnose, arabinose, xylose, mannose, glucose, and galactose; the most monosaccharide was glucose. The EPS hydrolyzed by TFA only detected three kinds of monosaccharides, including mannose, arabinose, and galactose, mainly mannose. The study has set a foundation for further analysis of monosaccharide composition and structure of EPS from O. sinensis