scholarly journals Pengaruh Jumlah Inokulum Sel Inang Bakteri E.coli BL21 (DE3) pLysS dan Waktu Overekspresi pada Produksi Protein Rekombinan Fim-C Salmonella typhi

2018 ◽  
Vol 4 (2) ◽  
pp. 98-106
Author(s):  
Muktiningsih - Nurjayadi ◽  
Izzatul Ilma Chairinnisa ◽  
Geta Putri Mentari ◽  
Dudi Hardianto ◽  
Asri Sulfianti ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Large Scale ◽  
Salmonella Typhi ◽  
Cell Number ◽  
Bacterial Cells ◽  
Typhoid Vaccine ◽  
Over Expression ◽  
Bacterial Cultures ◽  
Sds Page

Recombinant protein Fim-C S.typhi is a potential protein that can be used as an alternative typhoid vaccine and produced on a large scale. However, before entering into a large-scale stage, laboratory optimum data on the factors that affect the production process of Fim-C Salmonella typhi proteins are required. This study aims to obtain information regarding the effect of host cell number E.coli BL21 (DE3) pLysS and overexpression time on production of recombinant protein Fim-C Salmonella typhi as the basis for developing candidate of typhoid vaccine. The optimization process of overexpression was done by adding 0.5 mM IPTG (Isopropyl-β-D-thiogalactopyranoside) inducer into bacterial cultures of 2%, 4%, and 6% with 4, 5, and 6 hours over expression. The measurement of the concentration of Fim-C recombinant protein extracted samples were done by a spectrophotometric method used BCA Kit Assay Thermo ScientificTM with wavelength 590nm. The characterization of those protein extracts was performed using SDS-PAGE method. The results from the study concluded that the number of 4% E.coli bacterial cells and four-hour overexpression time are the optimum condition of Fim- C Salmonella typhi characterized by the presence of high-intensity bands at ± 31 kDa.  

scholarly journals BIOACTIVITY CHARACTERIZATION OF PURIFIED RECOMBINANT HYPOTHETICAL PROTEIN CODED BY OPEN READING FRAME-112 OF STREPTOMYCES.

2021 ◽  
Vol 52 (2) ◽  
pp. 502-511
Author(s):  
R. M. Al-Obaidi ◽  
G. F. Salih ◽  
B. F. Nore
Keyword(s):  
Protective Effect ◽  
Hypothetical Protein ◽  
Positive Control ◽  
Open Reading Frame ◽  
Bacterial Cells ◽  
Disk Diffusion Test ◽  
Reading Frame ◽  
Over Expression ◽  
Sds Page

This study was aimed to investigate the Open Reading Frame-112 (ORF-112) gene, which encoded for a hypothetical 218 amino acids protein in Streptomyces bacteria. A complete ORF-112 gene was synthesized, with addition of a 6xHis-Tag at the N-terminal location. The synthesized DNA nucleotides were sub-cloned into bacterial expression plasmid pBAT4. The pBAT4-ORF-112 plasmid transformed in bacterial cells BL21(De3)pLysS, intended for protein over expression, induced by isopropyl β- d-1-thiogalactopyranoside (IPTG). The IMAC affinity chromatography technique was deployed for protein purifications. Highly-purified fractions of ORF-112 were achieved by using affinity Ni++-columns. The purified ORF-112 protein was tested for possible biological activity.  The SDS-PAGE analysis exhibited a soluble 30 kDa size purified ORF-112 protein which showed a slight gel shifting from the predicted size. The virulent activity test on purified fractions of ORF-112 was measured using the Disk Diffusion Test and it disclosed a clear zone formed in response to fungi Candida albicans growth. The data implies that the ORF-112 protein has an acceptable protective effect against the fungus C. albicans as compared to positive control ketoconazole (KCZ) (P < 0.05) while the protein has a significantly lower protective effect against the fungus than Itraconazole (ICZ) (P > 0.05). The results clarify the hypothetical ORF-112 protein is a novel protein with protective response effect against fungi cells C. albicans on disk-diffusion.test.

scholarly journals Characterization of Polycaprolactone Nanohydroxyapatite Composites with Tunable Degradability Suitable for Indirect Printing

Polymers ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 295
Author(s):  
Stephanie E. Doyle ◽  
Lauren Henry ◽  
Ellen McGennisken ◽  
Carmine Onofrillo ◽  
Claudia Di Bella ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Large Scale ◽  
Three Dimensional ◽  
Cell Number ◽  
Compressive Modulus ◽  
Degradation Rates ◽  
Scaffold Materials ◽  
Natural Tissue ◽  
Complete Regeneration

Degradable bone implants are designed to foster the complete regeneration of natural tissue after large-scale loss trauma. Polycaprolactone (PCL) and hydroxyapatite (HA) composites are promising scaffold materials with superior mechanical and osteoinductive properties compared to the single materials. However, producing three-dimensional (3D) structures with high HA content as well as tuneable degradability remains a challenge. To address this issue and create homogeneously distributed PCL-nanoHA (nHA) scaffolds with tuneable degradation rates through both PCL molecular weight and nHA concentration, we conducted a detailed characterisation and comparison of a range of PCL-nHA composites across three molecular weight PCLs (14, 45, and 80 kDa) and with nHA content up to 30% w/w. In general, the addition of nHA results in an increase of viscosity for the PCL-nHA composites but has little effect on their compressive modulus. Importantly, we observe that the addition of nHA increases the rate of degradation compared to PCL alone. We show that the 45 and 80 kDa PCL-nHA groups can be fabricated via indirect 3D printing and have homogenously distributed nHA even after fabrication. Finally, the cytocompatibility of the composite materials is evaluated for the 45 and 80 kDa groups, with the results showing no significant change in cell number compared to the control. In conclusion, our analyses unveil several features that are crucial for processing the composite material into a tissue engineered implant.

Over-Expression of a Core Repeat from an Insect Silk Protein that forms Intramolecular Disulfide Bonds

1992 ◽  
Vol 292 ◽  
Author(s):  
Stanley V. Smith ◽  
Steven T. Case
Keyword(s):  
Recombinant Protein ◽  
Electrophoretic Mobility ◽  
Disulfide Bonds ◽  
Protein A ◽  
Reducing Agents ◽  
Silk Protein ◽  
Chironomus Tentans ◽  
Bacterial Cells ◽  
Gene Encoding ◽  
Over Expression

AbstractA gene encoding one complete [C+SR] core repeat from spIa, a 1000-kDa silk protein from Chironomus tentans, was synthesized and its recombinant protein expressed to high levels in bacterial cells. We observed that reducing agents significantly alter the electrophoretic mobility of this protein. A variety of data indicate that the purified recombinant protein is folded and its structure stabilized by two intramolecular disulfide bonds.

scholarly journals Purification of p24 protein expressed in Bacillus subtilis and evaluation of its immunogenicity in mice

2017 ◽  
Vol 1 (T1) ◽  
pp. 69-79 ◽  
Author(s):  
Tuom Thi Tinh Truong ◽  
Trang Thi Phuong Phan ◽  
Hoang Duc Nguyen
Keyword(s):  
Bacillus Subtilis ◽  
Recombinant Protein ◽  
Western Blot ◽  
Host Cell ◽  
Essential Role ◽  
Subtilis Strain ◽  
Total Proteins ◽  
Over Expression ◽  
Sds Page ◽  
P24 Protein

p24 protein is a component of the HIV particle capsid. It plays an essential role in HIV to infect into the host cell and in the cycle life of virus. Therefore, this protein can be used in the orientative study “to create and produce HIV’s vaccine”. This study created the new Bacillus subtilis strain which expressed p24 protein. B. subtilis a safety and non-toxic bacteria strain for humans and animals, has system expression to allow over expression recombinant protein up to 10-30 % of total proteins. Plasmid pHT1537 was cloned successfully, containing lysSN-6his-gagp24 gene to encode p24 protein fused with LysSN protein and to allow the expression of p24 protein in B. subtilis by IPTG inducer. The target protein in the cell was cheked by SDS-PAGE. The p24 fused protein was parified from His Trap column which contained Ni2+. Evaluation of the ability to produce antibody against p24 protein in mice by ELISA and Western blot was caried out.

scholarly journals PRODUCTION AND CHARACTERIZATION OF RECOMBINANT PROTEINS CONTAINING BET V 2 ANTIGENIC EPITOPES OF BETULA PENDULA

2012 ◽  
Vol 9 (6) ◽  
pp. 28-31
Author(s):  
A V Chernysheva ◽  
V V Tyutyaeva ◽  
A V Pivovarova ◽  
I V Andreev ◽  
M N Sankov ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Inclusion Bodies ◽  
Birch Pollen ◽  
Elisa Test ◽  
Recombinant Allergen ◽  
Bacterial Cells ◽  
E Coli ◽  
Immunological Tests ◽  
Linear Epitopes

Aim of investigation. Production of immunologically active recombinant protein of Bet v 2 allergen ofbirch pollen. Materials and methods. mRNA was isolated from a sample ofbirch pollen. cDNA library was derived using SMART technology. Gene Bet v 2 was amplified by means of PCR with primers from the cDNA. The resulting PCR fragment of the gene was cloned into the vector pET29b(+). The recombinant protein Bet v 2 was expressed in cells E. coli, transformed with a plasmid. The recombinant protein was purified using NiNTA agarose. Immunological activity of the recombinant protein Bet v 2 was measured by ELISA and immunoblot methods. Results. The production system of the recombinant allergen Bet v 2 preparation suitable for immunological tests was developed during the research project. In the first phase allergen Bet v 2 gene was cloned from birch pollen collected in Russia. The gene was inserted into the vector pET29(+) for expression in bacterial cells. The expression cell strain E. coli was obtained with this plasmid. The synthesis of the recombinant protein that accumulates in inclusion bodies was activated in bacterial cells. The procedure of recombinant protein Bet v 2 isolation from inclusion bodies was developed by one round of chromatography purification. The recombinant protein isolation was carried out by chromatography on Niagarose. The highly purified preparation of the recombinant allergen was obtained as a result. The recognition of the recombinant protein Bet v 2 by sera varied in ELISA, indicating a different degree of patients sensitization to this allergen. In the immunoblot test the preparation was active only in 15% of cases. Apparently, reactive epitopes of the allergen are mainly conformational ones and are active in ELISA test, whereas linear epitopes, that are active in immunoblot, are in the minority. Conclusion.The system for production of recombinant allergen Bet v 2 preparation suitable for immunological tests has been developed.

scholarly journals Cloning and Characterization of Aedes aegypti Trypsin Modulating Oostatic Factor (TMOF) Gut Receptor

Biomolecules ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 934
Author(s):  
Dov Borovsky ◽  
Kato Deckers ◽  
Anne Catherine Vanhove ◽  
Maud Verstraete ◽  
Pierre Rougé ◽  
...  
Keyword(s):  
Binding Sites ◽  
Protein Sequence ◽  
Bacterial Cells ◽  
E Coli ◽  
Trypsin Modulating Oostatic Factor ◽  
High Affinity ◽  
Sds Page ◽  
Atpase Inhibitors ◽  
Kinetics Of

Trypsin Modulating Oostatic Factor (TMOF) receptor was solubilized from the guts of female Ae. Aegypti and cross linked to His6-TMOF and purified by Ni affinity chromatography. SDS PAGE identified two protein bands (45 and 61 kDa). The bands were cut digested and analyzed using MS/MS identifying a protein sequence (1306 amino acids) in the genome of Ae. aegypti. The mRNA of the receptor was extracted, the cDNA sequenced and cloned into pTAC-MAT-2. E. coli SbmA− was transformed with the recombinant plasmid and the receptor was expressed in the inner membrane of the bacterial cell. The binding kinetics of TMOF-FITC was then followed showing that the cloned receptor exhibits high affinity to TMOF (KD = 113.7 ± 18 nM ± SEM and Bmax = 28.7 ± 1.8 pmol ± SEM). Incubation of TMOF-FITC with E. coli cells that express the receptor show that the receptor binds TMOF and imports it into the bacterial cells, indicating that in mosquitoes the receptor imports TMOF into the gut epithelial cells. A 3D modeling of the receptor indicates that the receptor has ATP binding sites and TMOF transport into recombinant E. coli cells is inhibited with ATPase inhibitors Na Arsenate and Na Azide.

scholarly journals Extraction of ADP-Heptose and Kdo2-Lipid A from E. coli Deficient in the Heptosyltransferase I Gene

2021 ◽  
Vol 11 (18) ◽  
pp. 8314
Author(s):  
Jozafina Milicaj ◽  
Colleen D. Castro ◽  
Nadiya Jaunbocus ◽  
Erika A. Taylor
Keyword(s):  
Bacterial Infections ◽  
Visual Cues ◽  
Large Scale ◽  
Lipid A ◽  
Biosynthetic Enzymes ◽  
Bacterial Cells ◽  
Site Specific ◽  
E Coli ◽  
Key Steps

The enzymes involved in lipopolysaccharide (LPS) biosynthesis, including Heptosyltransferase I (HepI), are critical for maintaining the integrity of the bacterial cell wall, and therefore these LPS biosynthetic enzymes are validated targets for drug discovery to treat Gram-negative bacterial infections. Enzymes involved in the biosynthesis of lipopolysaccharides (LPSs) utilize substrates that are synthetically complex, with numerous stereocenters and site-specific glycosylation patterns. Due to the relatively complex substrate structures, characterization of these enzymes has necessitated strategies to generate bacterial cells with gene disruptions to enable the extraction of these substrates from large scale bacterial growths. Like many LPS biosynthetic enzymes, Heptosyltransferase I binds two substrates: the sugar acceptor substrate, Kdo2-Lipid A, and the sugar donor substrate, ADP-l-glycero-d-manno-heptose (ADPH). HepI characterization experiments require copious amounts of Kdo2-Lipid A and ADPH, and unsuccessful extractions of these two substrates can lead to serious delays in collection of data. While there are papers and theses with protocols for extraction of these substrates, they are often missing small details essential to the success of the extraction. Herein detailed protocols are given for extraction of ADPH and Kdo2-Lipid A (KLA) from E. coli, which have had proven success in the Taylor lab. Key steps in the extraction of ADPH are clearing the extract through ultracentrifugation and keeping all water that touches anything in the extraction, including filters, at a pH of 8.0. Key steps in the extraction of KLA are properly lysing the dried down cells before starting the extraction, maximizing yield by allowing precipitate to form overnight, appropriately washing the pellet with phenol and dissolving the KLA in 1% TEA using visual cues, rather than a specific volume. These protocols led to increased yield and a higher success rate of extractions thereby enabling the characterization of HepI.

scholarly journals Production and bioactivity test of recombinant protein common carp growth hormone

2011 ◽  
Vol 10 (1) ◽  
pp. 44
Author(s):  
Deny Sapto Chondro Utomo ◽  
. Alimuddin ◽  
Agus Oman Sudrajat ◽  
Irvan Faizal
Keyword(s):  
Escherichia Coli ◽  
Growth Hormone ◽  
Body Weight ◽  
Recombinant Protein ◽  
Common Carp ◽  
Cyprinus Carpio ◽  
Bacterial Cells ◽  
Recombinant Growth Hormone ◽  
E Coli ◽  
Sds Page

<p>This study aimed to produce recombinant growth hormone <em>(r</em>GH) protein of common carp (<em>Cyprinus carpio</em>) and evaluate its bioactivity. DNA fragment encoding mature GH protein of common carp (<em>mCc</em>GH) was amplified by polymerase chain reaction (PCR) method and PCR products were then ligated into pCold I to generate pCold I-<em>mCc</em>GH protein expression vector. <em>Escherichia coli </em>BL21 (DE3) harboring pCold I-<em>mCc</em>GH was cultured in the 2xYT medium at 15 °C for 24 hours and protein production was induced by isopropyl-beta-thio galactopyranoside (IPTG). The inclusion bodies containing rGH protein from <em>E. coli </em>transformants were isolated by sonication method and analyzed by SDS-PAGE. The result showed that rGH with molecular weight of about 25 kDa was obtained. Common carp juveniles with average body weight of 5.2±0.4 g were intramuscularly injected once a week for 4 weeks with rGH protein solution from 1 μg bacterial cells per gram fish body weight. The result showed that juveniles fish injected with rGH grew 106.56% higher than control. This result indicated that rGH produced in <em>E. coli </em>BL21 possessed biological activity and it may be useful to improve growth of aquaculture species.</p> <p>Key words: growth hormone, recombinant protein, common carp</p> <p> </p> <p>ABSTRAK</p> <p>Penelitian ini bertujuan menghasilkan protein rekombinan hormon pertumbuhan (<em>growth hormone</em>, GH) dari ikan mas (<em>Cyprinus carpio</em>) dan menguji bioaktivitasnya. Fragmen DNA penyandi protein matang (<em>mature</em>) GH ikan mas (<em>mCc</em>GH) diamplifikasi dengan menggunakan metode PCR dan hasilnya kemudian diligasi ke dalam pCold-I untuk menghasilkan konstruksi vektor ekspresi pCold-I-<em>mCc</em>GH. Plasmid pCold-I-<em>mCc</em>GH ditransformasi ke bakteri <em>Escherichia coli</em> BL21 (DE3), dikultur dalam media 2xYT cair pada suhu 15°C selama 24 jam dan produksi protein diinduksi dengan menggunakan isopropyl-beta-thio galactopyranoside (IPTG). Badan inklusi yang mengandung protein rekombinan GH (rGH) dari bakteri <em>E. coli</em> transforman diisolasi menggunakan metode sonikasi dan dianalisis dengan menggunakan SDS-PAGE. Hasil penelitian menunjukkan bahwa rGH dengan bobot molekul sekitar 25 kDa berhasil diproduksi. Benih ikan mas dengan bobot rata-rata 5,15±0,4 g diinjeksi secara intramuskular satu kali per minggu selama 4 minggu dengan larutan rGH hasil ekstraksi dari 1 µg pelet bakteri/g bobot ikan. Benih yang disuntik dengan rGH tumbuh sekitar 100% lebih cepat bila dibandingkan dengan kontrol yang tidak diinjeksi rGH. Hasil ini mengindikasikan bahwa rGH yang diproduksi dalam bakteri <em>E. coli</em> memiliki bioaktivitas dan dapat bermanfaat untuk memacu pertumbuhan spesies ikan-ikan budidaya.</p> <p>Kata kunci: hormon pertumbuhan, protein rekombinan, ikan mas</p>

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