Immunocontraceptive potential of a GnRH receptor-based fusion recombinant protein

Abstract Background The management of stray dog population has been of utmost importance due to their overpopulation, increase in dog bites incidence, and rabies. Contraceptive vaccines, a non-surgical alternative to spaying and neutering are viewed as a valuable option for the management of dog population. In this study, the contraceptive potential of a recombinant fusion protein containing the three genes GnRH, GnRH receptor, and ZP3 was explored. Results The gene fragment encoding GnRH, GnRHR, and ZP3 along with the antigenic epitopes of canine distemper virus and tetanus toxoid was assembled, synthesized, and cloned into pET28a expression vector. The resulting construct GVAC08 was successfully transformed into BL21DE3 strain of E. coli and confirmed by colony PCR. The recombinant GVAC08 protein was expressed and purified using Ni-NTA and was confirmed to be a 50-KDa protein by SDS PAGE and Western blot. Mice were immunized with the GVAC08 protein using Freund’s complete adjuvant followed by a booster using Freund’s incomplete adjuvant. This induced a high antibody titer against GnRH, GnRH receptor, and ZP3 which was determined by ELISA. Conclusion Mating studies showed that the GVAC08 recombinant protein was able to reduce the litter size in immunized mice showing improved efficacy. However, the vaccine candidate with further improvements will be a viable contraceptive vaccine.

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Purification and reactivation of recombinant Synechococcus phytoene desaturase from an overexpressing strain of Escherichia coli

Biochemical Journal ◽

10.1042/bj2910687 ◽

1993 ◽

Vol 291 (3) ◽

pp. 687-692 ◽

Cited By ~ 27

Author(s):

P D Fraser ◽

H Linden ◽

G Sandmann

Keyword(s):

Escherichia Coli ◽

Recombinant Protein ◽

Deae Cellulose ◽

Cellular Protein ◽

Phytoene Desaturase ◽

E Coli ◽

Sds Page ◽

Purification Scheme ◽

Overexpressing Strain ◽

Cellulose Column

The Synechococcus phytoene desaturase has been isolated from an overexpressing strain of Escherichia coli. The plasma pPDSde135 mediated the overexpression of the full-length polypeptide directly. The recombinant protein comprised 5% of the total cellular protein and was found predominantly in the inclusion body fraction. Urea was used to solubilize the recombinant protein from the inclusion fraction and the protein was subsequently purified to homogeneity on a DEAE-cellulose column. The purification scheme yielded 4.0 mg of homogeneous desaturase protein after a 20-fold purification, recovering 40% of the original protein from a 100 ml suspension culture of E. coli. The recombinant desaturase had an apparent molecular mass of 53 kDa on SDS/PAGE and crossreacted with an antiserum raised against the expressed protein. Desaturase activity was restored upon the removal of urea. The enzyme catalysed the conversion of phytoene to zeta-carotene via phytofluene. These products of the desaturase reaction existed predominantly in a cis configuration. Lipid replenishment enhanced activity. NAD+ and NADP+ were observed to be involved, whilst FAD was an ineffective electron acceptor.

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Analytical and preparative biosynthesis of S100B recombinant protein using the pBT7-N-His vector system and preliminary studies of structure and function of this protein

Nauchno-prakticheskii zhurnal «Patogenez» ◽

10.25557/2310-0435.2018.04.161-164 ◽

2018 ◽

pp. 161-164

Author(s):

О.Л. Терёхина ◽

М.К. Нурбеков ◽

О.П. Дмитренко ◽

Д.М. Давыдов

Keyword(s):

Recombinant Protein ◽

Molecular Mechanisms ◽

The Body ◽

S100b Protein ◽

Vector System ◽

Diagnostic Systems ◽

E Coli ◽

Sds Page ◽

Bacterial Clone ◽

And Function

С целью исследований структуры и функций белка S100B в клетке и в тканях был проведен цикл работ по оптимизации экспрессии рекомбинантного белка (рекS100B) в E. coli. Проведены процедуры аналитической экспрессии рекS100B в составе рекомбинантной плазмиды pBT7-N-His-S100B03. При SDS-ПААГЭ лизатов клонов бактерий выявлена четко экспрессирующаяся полоса в 10 кДа, которая была идентифицирована как мономерная форма белка. Перспективы исследований рекS100B связаны с потенциальным его использованием для изучения тонких молекулярных механизмов PPI взаимодействий в системе S100B/RAGE рецептор как ключевого звена передачи сигналов в клетке и организме и в качестве перспективного объекта создания диагностических систем мониторинга состояний организма в норме и при патологии связанной с нарушениями регуляции гена и/или функций S100B белка. To study structure and functions of the S100B protein in cells and tissues, a series of studies was conducted to optimize the recombinant protein (recS100B) expression in E. coli. Procedures for analytical expression of recS100B in the pBT7-N-His-S100B03 recombinant plasmid were performed. In SDS-PAGE of bacterial clone lysate, a clear 10 kDa band expression was detected, which was identified as a monomeric form of the protein. Prospects for the S100B study are related with its potential use for investigating molecular mechanisms of PPI interactions in the S100B/RAGE system as a key signal transducer in the cell and body and as a promising object for developing diagnostic systems for monitoring the body state in normal and pathological conditions associated with impaired regulation of the gene and/or functions of the S100B protein.

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Effect of Ethanol and IPTG on the Recombinant Jembrana Trans-Activator of Transcriptation Protein Expression

Biosaintifika Journal of Biology & Biology Education ◽

10.15294/biosaintifika.v10i3.15596 ◽

2018 ◽

Vol 10 (3) ◽

pp. 559-564

Author(s):

Indriawati Indriawati ◽

Mega Salfia ◽

R Susanti ◽

Endang Tri Margawati

Keyword(s):

Recombinant Protein ◽

Protein Expression ◽

Experimental Research ◽

Quantitative Data ◽

Recombinant Protein Expression ◽

E Coli ◽

Qualitative And Quantitative ◽

Vaccine Product ◽

Sds Page ◽

Preliminary Study

Jembrana diseases are caused by Jembrana Diseases Virus (JDV). The previous study showed that Jembrana Trans-Activator of Trancriptation (JTAT) recombinant protein is effective as a vaccine for Jembrana diseases. The production of JTAT protein needs to be optimized to obtain a higher amount of vaccine. High expression of JTAT protein will produce a high vaccine product. This study aimed to examine the effect of the addition of ethanol and IPTG in E. coli media on the expression of JTAT recombinant protein. This research was experimental research with factorial RAL design with a variation factor of ethanol and IPTG. Qualitatively, the induction of each IPTG, ethanol and interaction between the two could induce the expression of JTAT protein and could be identified with SDS-PAGE at ±11.8 kDa. Statistically, the induction of IPTG, ethanol and interaction between the two were not significantly different. Qualitative and quantitative data show that ethanol can induce JTAT protein expression. This result can be used as a preliminary study to test the effectiveness of ethanol as a substitute for IPTG in inducing the recombinant protein expression.

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Production and bioactivity test of recombinant protein common carp growth hormone

Jurnal Akuakultur Indonesia ◽

10.19027/jai.10.44-50 ◽

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

This study aimed to produce recombinant growth hormone (rGH) protein of common carp (Cyprinus carpio) and evaluate its bioactivity. DNA fragment encoding mature GH protein of common carp (mCcGH) was amplified by polymerase chain reaction (PCR) method and PCR products were then ligated into pCold I to generate pCold I-mCcGH protein expression vector. Escherichia coli BL21 (DE3) harboring pCold I-mCcGH 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 E. coli 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 E. coli BL21 possessed biological activity and it may be useful to improve growth of aquaculture species. Key words: growth hormone, recombinant protein, common carp ABSTRAK Penelitian ini bertujuan menghasilkan protein rekombinan hormon pertumbuhan (growth hormone, GH) dari ikan mas (Cyprinus carpio) dan menguji bioaktivitasnya. Fragmen DNA penyandi protein matang (mature) GH ikan mas (mCcGH) diamplifikasi dengan menggunakan metode PCR dan hasilnya kemudian diligasi ke dalam pCold-I untuk menghasilkan konstruksi vektor ekspresi pCold-I-mCcGH. Plasmid pCold-I-mCcGH ditransformasi ke bakteri Escherichia coli 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 E. coli 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 E. coli memiliki bioaktivitas dan dapat bermanfaat untuk memacu pertumbuhan spesies ikan-ikan budidaya. Kata kunci: hormon pertumbuhan, protein rekombinan, ikan mas

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Expression of Hepatitis B viral surface antigen (Pre S1) in E. coli

International Journal of Research in Pharmaceutical Sciences ◽

10.26452/ijrps.v11i3.2406 ◽

2020 ◽

Vol 11 (3) ◽

pp. 3046-3052

Author(s):

Shahad Basel Ismail ◽

Yaseen Ismael Mamoori ◽

Ibrahim Abdulla Ahmed

Keyword(s):

Molecular Weight ◽

Hepatitis B ◽

Protein Band ◽

Effective Vaccine ◽

Chronic Hepatitis B Infection ◽

Colony Pcr ◽

E Coli ◽

Sds Page ◽

B Virus ◽

Viral Surface

Hepatitis B is the most common liver diseases, which caused by hepatitis B virus (HBV) infection. There are around 257 million people around the world suffer from severe chronic hepatitis B infection. Therefore, it is necessary to develop a vaccine to prevent viral infection. PreS1 is one of the HBV envelope proteins that have been proved to be an effective vaccine. Accordingly, Viral DNA was purified from patients’ serums and amplified by PCR using specific primers. Amplicons of 324 bp bands of PreS1 was observed on gel electrophoresis. The PreS1 was cloned into pTXB21 plasmid to form the recombinant plasmid pTXB1_PreS1 and transformed into DH5α E. coli. Screening of transformants was done using Colony PCR and Sequencing. Alignment of 26 polypeptide sequences showed conservation of this region. The pTXB1_PreS1 was retransformed into T7 Express Competent E. coli and screened using colony PCR. The PreS1 was expressed as a recombinant protein fused to an intein tag with a molecular weight of ~ 39.5 kD. The PreS1 protein was purified by a single affinity chromatography step and after cleaved from intein tag by Dithiothreitol the obtained protein had a molecular weight of ~ 11.5 kD. Only one protein band was observed on the SDS-page gel. The PreS1 protein was successfully cloned and expressed in E. coli, which can be used as a vaccine against HBV.

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Improving the Recombinant Protein Expression of Human Galectin-3 in BL21 Bacterial Host System

Journal of Pharmaceutical Research International ◽

10.9734/jpri/2020/v32i3430970 ◽

2020 ◽

pp. 111-115

Author(s):

Praveen Kumar Vemuri ◽

Nikhil Reddy Varakala ◽

Divyanshu Dhakate ◽

Tripura Ravavarapu ◽

Faina Philberta Dumpala ◽

...

Keyword(s):

Recombinant Protein ◽

Bacterial Culture ◽

Recombinant Protein Expression ◽

Shuttle Vector ◽

Bacterial Host ◽

E Coli ◽

His Tag ◽

Sds Page ◽

Galectin 3 ◽

Characterization Studies

Background: Regardless of the broad explore in the territory of glycobiology concerning structure and capacity of glycans, lectins and glycosylation forms, numerous viewpoints are still left unexplored. Aim: In this study, we analyzed the effect of shuttle vector on the secretion of human galectin recombinant protein. Methods: The galectin was expressed in E. coli BL21 by growing the bacterial culture in SOC medium and purified by nickel-based affinity chromatography due to its His-tag. Results: After cell lysis the protein was identified as a single 29 KDa band by 12% SDS-PAGE. Conclusion: Characterization studies clearly revealed that the purified protein was indeed galectin 3.

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Design, Expression and Purification of Strongyloides stercoralis IgG4 Immunoreactive Protein (NIE) in Escherichia coli

Iranian Journal of Parasitology ◽

10.18502/ijpa.v15i3.4198 ◽

2020 ◽

Author(s):

Katayoun DASTAN ◽

Mehdi ASSMAR ◽

Nour AMIRMOZAFARI ◽

Fariborz Mansour GHANAEI ◽

Mirsasan MIRPOUR

Keyword(s):

Escherichia Coli ◽

Recombinant Protein ◽

Western Blotting ◽

Expression System ◽

Strongyloides Stercoralis ◽

Health Concern ◽

Immunoreactive Protein ◽

E Coli ◽

Elisa Kit ◽

Sds Page

Background: Strongyloidiasis is a public health concern in northern regions of Iran, caused by Strongyloides stercoralis. Auto-infection cycle can be resulted in high parasitic load, especially in immunocompromised hosts. Because of low sensitivity of stool culture and stool-based microscopy techniques, detection of antibodies in patient’s sera can be an alternative diagnostic technique for detection of the nematode. In the present study, as the first step of the development of an ELISA kit for the detection of antibodies against the nematode, IgG4 immunoreactive protein (NIE) was expressed in Escherichia coli expression system, purified and verified. Methods: The NIE gene sequence was retrieved from the GenBank. This sequence was codon-optimized for the expression in E. coli BL21 (DE3). The sequence was inserted into the expression vector pET-30b (+). The recombinant vector was then transferred into competent E. coli BL21 (DE3). Transformed colonies were selected and verified by colony PCR. NIE gene expression was induced with IPTG induction. The protein production was evaluated by SDS-PAGE and verified using Western blotting. Results: The codon-optimized NIE gene had required parameters for expression in E. coli. NIE protein was proved and verified by SDS-PAGE and Western blotting. Conclusion: NIE recombinant protein was successfully expressed in E. coli expression system in appropriate amounts. The recombinant protein can be used for developing ELISA kit in diagnosis of S. stercoralis.

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EXPRESSION AND PURIFICATION OF RECOMBINANT T4 DNA LIGASE IN E. COLI

Science and Technology Development Journal ◽

10.32508/stdj.v14i3.1991 ◽

2011 ◽

Vol 14 (3) ◽

pp. 73-79

Author(s):

Duy Long Duong ◽

Duc Van Luong ◽

Hieu Thi Phuong Nguyen ◽

Hoa Thanh Tran ◽

Thao Thi Phuong Dang ◽

...

Keyword(s):

Recombinant Protein ◽

Western Blot ◽

Target Protein ◽

Dna Ligase ◽

Expression And Purification ◽

T4 Dna Ligase ◽

E Coli ◽

Sds Page

In this study, we report results on the expression and purification of recombinant T4 DNA ligase. Plasmid pET16b-T4Dnl contains the gp30 gene which encodes for T4 DNA ligase. The target protein is fused with 10xHis tag to facilitate the purification and recovery. pET16b-T4Dnl was transformed into E. coli BL21(DE3) and then induced the expression of 10xHis-T4Dnl by IPTG. The recombinant protein was purified by Ni-NTA chromatography and confirmed by SDS-PAGE and Western blot. The activity of purified protein was tested by joining DNA λ/HindIII.

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Enhanced Biocatalytic Activity of Recombinant Lipase Immobilized on Gold Nanoparticles

Current Pharmaceutical Biotechnology ◽

10.2174/1389201020666190416144650 ◽

2019 ◽

Vol 20 (6) ◽

pp. 497-505 ◽

Cited By ~ 1

Author(s):

Abeer M. Abd El-Aziz ◽

Mohamed A. Shaker ◽

Mona I. Shaaban

Keyword(s):

Gold Nanoparticles ◽

Lipolytic Activity ◽

Market Demand ◽

Biotechnological Applications ◽

Lipase Gene ◽

Expression Plasmid ◽

E Coli ◽

Nickel Affinity Chromatography ◽

Recombinant Production ◽

Sds Page

Background: Bacterial lipases especially Pseudomonas lipases are extensively used for different biotechnological applications. Objectives: With the better understanding and progressive needs for improving its activity in accordance with the growing market demand, we aimed in this study to improve the recombinant production and biocatalytic activity of lipases via surface conjugation on gold nanoparticles. Methods: The full length coding sequences of lipase gene (lipA), lipase specific foldase gene (lipf) and dual cassette (lipAf) gene were amplified from the genomic DNA of Pseudomonas aeruginosa PA14 and cloned into the bacterial expression vector pRSET-B. Recombinant lipases were expressed in E. coli BL-21 (DE3) pLysS then purified using nickel affinity chromatography and the protein identity was confirmed using SDS-PAGE and Western blot analysis. The purified recombinant lipases were immobilized through surface conjugation with gold nanoparticles and enzymatic activity was colorimetrically quantified. Results: Here, two single expression plasmid systems pRSET-B-lipA and pRSET-B-lipf and one dual cassette expression plasmid system pRSET-B-lipAf were successfully constructed. The lipolytic activities of recombinant lipases LipA, Lipf and LipAf were 4870, 426 and 6740 IUmg-1, respectively. However, upon immobilization of these recombinant lipases on prepared gold nanoparticles (GNPs), the activities were 7417, 822 and 13035 IUmg-1, for LipA-GNPs, Lipf-GNPs and LipAf-GNPs, respectively. The activities after immobilization have been increased 1.52 and 1.93 -fold for LipA and LipAf, respectively. Conclusion: The lipolytic activity of recombinant lipases in the bioconjugate was significantly increased relative to the free recombinant enzyme where immobilization had made the enzyme attain its optimum performance.

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Multiple Microfermentor Battery: a Versatile Tool for Use with Automated Parallel Cultures of Microorganisms Producing Recombinant Proteins and for Optimization of Cultivation Protocols

Applied and Environmental Microbiology ◽

10.1128/aem.00239-06 ◽

2006 ◽

Vol 72 (8) ◽

pp. 5225-5231 ◽

Cited By ~ 21

Author(s):

Emmanuel Frachon ◽

Vincent Bondet ◽

Hélène Munier-Lehmann ◽

Jacques Bellalou

Keyword(s):

Recombinant Protein ◽

Recombinant Proteins ◽

Protein Production ◽

Batch Cultures ◽

E Coli ◽

Working Volume ◽

Versatile Tool ◽

Labview Program ◽

Medium Formulation ◽

Conventional Medium

ABSTRACT A multiple microfermentor battery was designed for high-throughput recombinant protein production in Escherichia coli. This novel system comprises eight aerated glass reactors with a working volume of 80 ml and a moving external optical sensor for measuring optical densities at 600 nm (OD600) ranging from 0.05 to 100 online. Each reactor can be fitted with miniature probes to monitor temperature, dissolved oxygen (DO), and pH. Independent temperature regulation for each vessel is obtained with heating/cooling Peltier devices. Data from pH, DO, and turbidity sensors are collected on a FieldPoint (National Instruments) I/O interface and are processed and recorded by a LabVIEW program on a personal computer, which enables feedback control of the culture parameters. A high-density medium formulation was designed, which enabled us to grow E. coli to OD600 up to 100 in batch cultures with oxygen-enriched aeration. Accordingly, the biomass and the amount of recombinant protein produced in a 70-ml culture were at least equivalent to the biomass and the amount of recombinant protein obtained in a Fernbach flask with 1 liter of conventional medium. Thus, the microfermentor battery appears to be well suited for automated parallel cultures and process optimization, such as that needed for structural genomics projects.

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