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

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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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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EXPRESSION OF CELLULOSE-DEGRADING ENDOGLUCANASE FROM BACILLUS SUBTILIS USING PTOLT EXPRESSION SYSTEM IN ESCHERICHIA COLI

Cellulose Chemistry and Technology ◽

10.35812/cellulosechemtechnol.2021.55.50 ◽

2021 ◽

Vol 55 (5-6) ◽

pp. 619-627

Author(s):

HÜLYA KUDUĞ CEYLAN ◽

YAKUP ULUSU ◽

SEMA BILGIN ◽

İSA GÖKÇE

Keyword(s):

Escherichia Coli ◽

Bacillus Subtilis ◽

Expression System ◽

Industrial Applications ◽

Enzyme Analysis ◽

Restriction Enzyme Analysis ◽

E Coli ◽

Glycosidic Bonds ◽

Sds Page ◽

Dna Fragment

Endoglucanases randomly hydrolyse the cellulose chains by acting upon internal β-1,4-D-glycosidic bonds and are used extensively in industrial applications. In this study, bacterial endoglucanase gene yhfE was obtained by PCR, using primers based on genomic sequences of Bacillus subtilis strains. 1041 bp DNA fragment of yhfE was cloned into Escherichia coli DH5α through the use of pTolT expression plasmid. PCR, restriction enzyme analysis and DNA sequencing were performed in order to confirm the cloning. E. coli BL21-AI cells expressed the yhfE after induction at 0.04% of arabinose concentration for 4 h. The expected 38.7 kDa size yhfE protein after digestion with thrombin of the His-tagged fusion protein (yhfE-TolAIII) was visualized by SDS-PAGE. The yhfE-TolAIII production yield was approximately 82 mg/L. The recombinant yhfE was characterized by MALDI-TOF mass spectrometry and CD analysis.

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Impact of the Expression System on Recombinant Protein Production in Escherichia coli BL21

Frontiers in Microbiology ◽

10.3389/fmicb.2021.682001 ◽

2021 ◽

Vol 12 ◽

Author(s):

Gema Lozano Terol ◽

Julia Gallego-Jara ◽

Rosa Alba Sola Martínez ◽

Adrián Martínez Vivancos ◽

Manuel Cánovas Díaz ◽

...

Keyword(s):

Escherichia Coli ◽

Recombinant Protein ◽

Protein Expression ◽

Recombinant Proteins ◽

Copy Number ◽

Recombinant Protein Production ◽

Protein Production ◽

Recombinant Protein Expression ◽

Expression System ◽

E Coli

Recombinant protein production for medical, academic, or industrial applications is essential for our current life. Recombinant proteins are obtained mainly through microbial fermentation, with Escherichia coli being the host most used. In spite of that, some problems are associated with the production of recombinant proteins in E. coli, such as the formation of inclusion bodies, the metabolic burden, or the inefficient translocation/transport system of expressed proteins. Optimizing transcription of heterologous genes is essential to avoid these drawbacks and develop competitive biotechnological processes. Here, expression of YFP reporter protein is evaluated under the control of four promoters of different strength (PT7lac, Ptrc, Ptac, and PBAD) and two different replication origins (high copy number pMB1′ and low copy number p15A). In addition, the study has been carried out with the E. coli BL21 wt and the ackA mutant strain growing in a rich medium with glucose or glycerol as carbon sources. Results showed that metabolic burden associated with transcription and translation of foreign genes involves a decrease in recombinant protein expression. It is necessary to find a balance between plasmid copy number and promoter strength to maximize soluble recombinant protein expression. The results obtained represent an important advance on the most suitable expression system to improve both the quantity and quality of recombinant proteins in bioproduction engineering.

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Escherichia coli σ70 promoters allow expression rate control at the cellular level in genome-integrated expression systems

10.21203/rs.2.19057/v2 ◽

2020 ◽

Author(s):

Artur Schuller ◽

Monika Cserjan-Puschmann ◽

Christopher Tauer ◽

Johanna Jarmer ◽

Martin Wagenknecht ◽

...

Keyword(s):

Escherichia Coli ◽

Recombinant Protein ◽

Recombinant Protein Production ◽

Protein Production ◽

Expression System ◽

Cellular Level ◽

Lac Repressor ◽

Expression Systems ◽

E Coli ◽

Basal Expression

Abstract Background The genome-integrated T7 expression system offers significant advantages, in terms of productivity and product quality, even when expressing the gene of interest (GOI) from a single copy of. Compared to plasmid-based expression systems, this system does not incur a plasmid-mediated metabolic load, and it does not vary the dosage of the GOI during the production process. However, long-term production with T7 expression system leads to a rapidly growing non-producing population, because the T7 RNA polymerase (RNAP) is prone to mutations. The present study aimed to investigate whether two σ 70 promoters, which were recognized by the Escherichia coli host RNAP, might be suitable in genome-integrated expression systems. We applied a promoter engineering strategy that allowed control of expressing the model protein, GFP, by introducing lac operators ( lacO ) into the constitutive T5 and A1 promoter sequences. Results We showed that, in genome-integrated E. coli expression systems that used σ 70 promoters, the number of lacO sites must be well balanced. Promoters containing three and two lacO sites exhibited low basal expression, but resulted in a complete stop in recombinant protein production in partially induced cultures. In contrast, expression systems regulated by a single lacO site and the lac repressor element, lacI Q , on the same chromosome caused very low basal expression, were highly efficient in recombinant protein production, and enables fine-tuning of gene expression levels on a cellular level. Conclusions Based on our results, we hypothesized that this phenomenon was associated with the autoregulation of the lac repressor protein, LacI. We reasoned that the affinity of LacI for the lacO sites of the GOI must be lower than the affinity of LacI to the lacO sites of the endogenous lac operon; otherwise, LacI autoregulation could not take place, and the lack of LacI autoregulation would lead to a disturbance in lac repressor-mediated regulation of transcription. By exploiting the mechanism of LacI autoregulation, we created a novel E. coli expression system for use in recombinant protein production, synthetic biology, and metabolic engineering applications.

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Escherichia coli σ70 promoters allow expression rate control at the cellular level in genome-integrated expression systems

10.21203/rs.2.19057/v1 ◽

2019 ◽

Author(s):

Artur Schuller ◽

Monika Cserjan-Puschmann ◽

Christopher Tauer ◽

Johanna Jarmer ◽

Martin Wagenknecht ◽

...

Keyword(s):

Escherichia Coli ◽

Recombinant Protein ◽

Recombinant Protein Production ◽

Protein Production ◽

Expression System ◽

Lac Repressor ◽

Expression Systems ◽

E Coli ◽

Basal Expression ◽

T7 Expression System

Abstract Background The genome-integrated T7 expression system offers significant advantages, in terms of productivity and product quality, even when expressing the gene of interest (GOI) from a single copy of. Compared to plasmid-based expression systems, this system does not incur a plasmid-mediated metabolic load, and it does not vary the dosage of the GOI during the production process. However, long-term production with T7 expression system leads to a rapidly growing non-producing population, because the T7 RNA polymerase (RNAP) is prone to mutations. The present study aimed to investigate whether two σ 70 promoters, which were recognized by the Escherichia coli host RNAP, might be suitable in genome-integrated expression systems. We applied a promoter engineering strategy that allowed control of expressing the model protein, GFP, by introducing lac operators ( lacO ) into the constitutive T5 and A1 promoter sequences.Results We showed that, in genome-integrated E. coli expression systems that used σ 70 promoters, the number of lacO sites must be well balanced. Promoters containing three and two lacO sites exhibited low basal expression, but resulted in a complete stop in recombinant protein production in partially induced cultures. In contrast, expression systems regulated by a single lacO site and the lac repressor element, lacI Q , on the same chromosome caused very low basal expression, were highly efficient in recombinant protein production, and enables fine-tuning of gene expression levels on a cellular level.Conclusions Based on our results, we hypothesized that this phenomenon was associated with the autoregulation of the lac repressor protein, LacI. We reasoned that the affinity of LacI for the lacO sites of the GOI must be lower than the affinity of LacI to the lacO sites of the endogenous lac operon; otherwise, LacI autoregulation could not take place, and the lack of LacI autoregulation would lead to a disturbance in lac repressor-mediated regulation of transcription. By exploiting the mechanism of LacI autoregulation, we created a novel E. coli expression system for use in recombinant protein production, synthetic biology, and metabolic engineering applications.

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Escherichia coli BL21(DE3) expression system using TorA signal peptide for Recombinant Human Albumin (rHA) secretion

International Journal of Research in Pharmaceutical Sciences ◽

10.26452/ijrps.v10i4.1640 ◽

2019 ◽

Vol 10 (4) ◽

pp. 3319-3324 ◽

Cited By ~ 1

Author(s):

Iman P. Maksum ◽

Astri Lestari ◽

Retna P. Fauzia ◽

Saadah D. Rachman ◽

Ukun M.S. Soedjanaatmadja

Keyword(s):

Escherichia Coli ◽

Molecular Weight ◽

Signal Peptide ◽

Recombinant Dna ◽

Expression System ◽

Foreign Protein ◽

Competent Cell ◽

Promising Alternative ◽

E Coli ◽

Sds Page

Human serum albumin (HSA) is the most abundant protein in blood plasm. This protein consisted of 585 amino acids with a molecular weight of 66 kDa and 17 disulfide bonds. HSA obtained from conventional technique allow viral or prion contamination. For that reason, recombinant DNA technology becomes a promising alternative. Because of its well-known genetic, simplicity, and capacity to accommodate many foreign protein, Escherichia coli remains the most widely used in the production of recombinant proteins. But, overproduction of protein may lead to the formation of inclusion bodies and proteolytic degradation. These problems can be overcome by using protease-deficient strain and protein secretion into periplasmic space. The objective of this research is to secrete recombinant HA on E. coli BL21(DE3) using TorA signal peptide and proved using SDS-PAGE. This research method begins with the preparation of competent cell and transformation of E. coli BL21(DE3), expression of recombinant HA in E. coli BL21(DE3), and characterization of expression result by using SDS-PAGE. The result of this study was rHSA can be secreted into extracellular medium using TorA signal peptide with a molecular weight of ± 66.5 kDa.

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An assay of human tyrosine protein kinase ABL activity using an Escherichia coli protein expression system

BioTechniques ◽

10.2144/btn-2020-0154 ◽

2021 ◽

Author(s):

Emiko Kinoshita-Kikuta ◽

Momoka Yoshimoto ◽

Marina Yano ◽

Eiji Kinoshita ◽

Tohru Koike

Keyword(s):

Escherichia Coli ◽

Protein Kinase ◽

Expression System ◽

Kinase Domain ◽

Wild Type ◽

E Coli ◽

Abl Kinase ◽

Sds Page ◽

Tyrosine Protein Kinase ◽

Comparative Results

ABL, a human tyrosine protein kinase, and its substrate are co-expressed in Escherichia coli. Tyrosine phosphorylation of the substrate in E. coli was detected using Phos-tag SDS-PAGE. The bacterial co-expression system was used as a field for the kinase reaction to evaluate the enzymatic activity of five types of ABL kinase domain mutants. Relative to wild-type ABL, kinase activity was comparable in the H396P mutant, reduced in both Y253F and E255K mutants and undetectable in T315I and M351T mutants. These comparative results demonstrated that the phosphorylation states of the mutants correlated with their activity. The bacterial co-expression system permits rapid production of tyrosine kinase variants and provides a simple approach for examining their structure–activity relationships.

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CLONAGEM, EXPRESSÃO E CARACTERIZAÇÃO DA NUCLEOPROTEÍNA RECOMBINANTE DO VÍRUS DA BRONQUITE INFECCIOSA EM ESCHERICHIA COLI E EM PICHIA PASTORIS

Arquivos do Instituto Biológico ◽

10.1590/1808-1657v77p0012010 ◽

2010 ◽

Vol 77 (1) ◽

pp. 1-9

Author(s):

A.M. Gibertoni ◽

M.C.M. Gonçalves ◽

M.F.S. Montassier ◽

C.C. Fernandes ◽

H.J. Montassier

Keyword(s):

Escherichia Coli ◽

Pichia Pastoris ◽

Western Blotting ◽

Rt Pcr ◽

E Coli ◽

Sds Page

RESUMO O gene da proteína de nucleocapsídeo (1.230 pb) da estirpe M41 do vírus da bronquite infecciosa (VBI) foi amplificado pelas reações de transcrição reversa e em cadeia da polimerase (RT-PCR) e clonado, em seguida, em dois sistemas; pET28a - Escherichia coli e pFLD -Pichia pastoris. Os produtos recombinantes construídos para expressão (pET28a-N ou pFLD-N) foram identificados por análises de PCR e de sequenciamento de nucleotídeos. Os clones transformantes da linhagem BL21 de E. coli e da linhagem GS115 de P. pastoris foram submetidos aos protocolos apropriados de indução. A expressão da proteína N de fusão com etiqueta de poli-histidina e com massa molecular de 54 kDa foi determinada pelas técnicas de SDS-PAGE e de Western blotting, confirmando-se que ambas proteínas N recombinantes apresentaram tamanhos e antigenicidade compatíveis com a proteína N nativa do próprio VBI. O sistema E. coli expressou uma quantidade relevante da proteína N recombinante, enquanto que o sistema P. pastoris produziu uma baixa recuperação dessa proteína recombinante. A proteína N recombinante gerada pelo sistema bacteriano foi purificada em resina de níquel-sepharose. O conjunto de resultados indica que o sistema de expressão constituído por pET28a – E. coli é mais efetivo para produzir a proteína N recombinante do VBI destinada ao uso como antígeno para detectar anticorpos anti-virais específicos em ensaios de imunodiagnóstico para essa infecção viral.

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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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Cloning and Characterization of SpcrtR and Its Expression in Escherichia Coli for Zeaxanthin Production

10.21203/rs.3.rs-543628/v1 ◽

2021 ◽

Author(s):

Jun Ma ◽

Yuxi Yin ◽

Chenqiang Qian ◽

Jiaxin Chen ◽

Di-Feng Ren

Keyword(s):

Western Blotting ◽

High Performance ◽

Expression System ◽

Specific Primers ◽

E Coli ◽

Sds Page ◽

Clone And Express ◽

Rate Limiting ◽

Β Carotene

Abstract Zeaxanthin is produced by a series of enzyme catalytic reactions. β-carotene hydroxylase is a key rate-limiting enzyme that catalyzes the conversion of β-carotene to zeaxanthin. The purpose of this study was to clone and express the Spirulina platensis β-carotene hydroxylase gene (SpcrtR) in E. coli. SpcrtR was amplified using specific primers and cloned in vector pGEX-6p-1, the SpcrtR protein (35kDa) was expressed from pGEX-6p-1-SpcrtR in E. coli. Gene expression was analyzed by SDS-PAGE and western blotting. The accumulation of carotenoids was detected by high-performance liquid chromatography (HPLC). SDS-PAGE and western blotting analysis confirmed that SpcrtR protein (35kDa) was expressed in E. coli expression system. Further, the HPLC results demonstrated that SpcrtR can partially catalyze β-carotene to produce zeaxanthin in E. coli. Zeaxanthin and β-carotene yields in recombinant E.coli were 85.71% (±0.4%) and 14.7% (±0.6%), respectively. The results in our study verified that SpCRTR enzyme partially catalyzes the substrate β-carotene to form zeaxanthin. Overall, SpCRTR provides a new choice for enzymatic zeaxanthin production.

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