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

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.

scholarly journals CONSTRUCTION, EXPRESSION AND PURIFICATION OF RECOMBINANT PRE-MATURE PEPTIDE OF PLANTARICIN F FROM Lactobacillus plantarum S34 IN Escherichia coli

2015 ◽  
Vol 16 (1) ◽  
pp. 31
Author(s):  
Kusdianawati Kusdianawati ◽  
Apon Zaenal Mustopa ◽  
Suharsono Suharsono ◽  
Bugi Ratno Budiarto ◽  
Fatimah Fatimah ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Lactobacillus Plantarum ◽  
Proteolytic Enzymes ◽  
Leader Peptide ◽  
Human Consumption ◽  
Mature Peptide ◽  
Expression And Purification ◽  
Food Preservative ◽  
E Coli ◽  
Sds Page

Plantaricin is one of bacteriocins that have the potential to be used as food preservative. Plantaricin is safe for human consumption because it can be easily degraded by proteolytic enzymes. The objective of this study was to express and purify recombinant pre-mature peptide of plantaricin F from <em>Lactobacillus plantarum</em> S34 in <em>Escherichia coli</em>. Plantaricin gene-specific primer was used to obtain pln F structural gene amplicon from L. <em>plantarum</em> S34. This amplicon was cloned in pET32a vector and expressed in E. coli BL21 (DE3) pLysS. Pre-mature plantaricin F peptide was expressed as Histagged-fusion protein and separated by Co2+-chelating affinity chromatography. L. <em>plantarum</em> S34-derived pre-mature plantaricin F peptide fused with thioredoxin-(His)6tag had successfully been expressed in E. <em>coli</em> BL21 (DE3) pLysS using pET32a as an expression vector. The fused recombinant pln F as pre-mature state expressed had a molecular mass of +24 kDa, meanwhile the fused recombinant that contained only the leader peptide of pln F appeared as +20 kDa based on SDS-PAGE separations. The optimal production of fused recombinant pln F as soluble fraction was obtained when culture condition was added with 0.5 mM of IPTG and incubated at 22°C for 5 hours (OD~1). Furthermore, the expression of fused recombinant pln F as its pre-mature peptide pointed out that the pln F’s leader peptide could be proteolytically cleaved by a system in heterologous cells. Overall, heterologous pln F production as pre-mature peptide fused with thioredoxin-(His)6tag had been well established. From this research, we expect plantaricin F can be expressed and purified in E. coli.

Cloning, expression, and purification of HpaA-CagA fusion recombinant protein of Helicobacter pylori in E. coli BL 21 strain

Gene Reports ◽  
2019 ◽  
Vol 16 ◽  
pp. 100417 ◽  
Author(s):  
Abbas Shapouri Moghaddam ◽  
Kiarash Ghazvini ◽  
Abbas Bahador ◽  
Mohammad Derakhshan ◽  
Azad Khaledi
Keyword(s):  
Helicobacter Pylori ◽  
Recombinant Protein ◽  
Expression And Purification ◽  
E Coli

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.

Heterologous Expression of SARS-CoV ORF10 and X5 Genes in E. coli and Streptomyces lividans TK24

2007 ◽  
Vol 62 (9-10) ◽  
pp. 765-771
Author(s):  
Jiang-qiang Kong ◽  
Wei Wang ◽  
Guan-hua Du ◽  
Ping Zhu ◽  
Ke-di Cheng
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Western Blot ◽  
Heterologous Expression ◽  
Streptomyces Lividans ◽  
Potential Functions ◽  
Structural Genes ◽  
First Report ◽  
E Coli ◽  
Accessory Genes ◽  
Sds Page

In previous studies a variety of novel accessory genes has been identified that were interspersed among the structural genes of the SARS-CoV (severe acute respiratory syndrome coronavirus) genome. The predicted unknown proteins (PUPs) encoded by the accessory genes, which are considered to be unique to the SARS-CoV genome, might play important roles in the SARS-CoV infection. Two of these genes, called ORF10 and X5, were synthesized and introduced into E. coli and Streptomyces lividans TK24, respectively. SDS-PAGE and Western blot revealed that the ORF10 and X5 genes have been expressed in the two hosts. This is the first report of heterologous expression of ORF10 and X5 genes in E. coli and S. lividans TK24. This work makes it possible to study the structure and potential functions of proteins encoding by these two genes.

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

1993 ◽  
Vol 291 (3) ◽  
pp. 687-692 ◽  
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.

scholarly journals Optimization of Expression, Purification and Secretion of Functional Recombinant Human Growth Hormone in Prokaryotic Hosts Using Modified Staphylococcal Protein A Signal Peptide

2021 ◽  
Author(s):  
Garshasb Rigi ◽  
Amin Rostami ◽  
Habib Ghomi ◽  
Gholamreza Ahmadian ◽  
Vasiqe Sadat Mirbagheri ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Western Blot ◽  
Signal Peptide ◽  
Extracellular Space ◽  
Culture Medium ◽  
Active Form ◽  
Protein A ◽  
Human Growth ◽  
E Coli ◽  
Sds Page

Abstract Background: Human Growth Hormone (hGH) is a glycoprotein released from the pituitary gland. Due to the wide range of effects in humans, any disruption in hGH secretion could have serious consequences. This highlights the clinical importance of hGH production in the treatment of different diseases associated with a deficiency of this hormone. The production of recombinant mature hormone in suitable hosts and secretion of this therapeutic protein into the extracellular space can be considered as one of the best cost-effective approaches not only to obtain the active form of the protein but also endotoxin-free preparation. Since the natural growth hormone signal peptide is of eukaryotic origin and is not detectable by any of the E. coli secretory systems, including Sec and Tat, and is therefore unable to secrete hGH in the prokaryotic systems, designing a new and efficient signal peptide is essential to direct hGh to the extracellular space. Results: In this study, using a combination of the bioinformatics design and molecular genetics, the protein A signal peptide from Staphylococcus aureus was modified, redesigned and then fused to the mature hGH coding region. The recombinant hGH was then expressed in E. coli and successfully secreted to the medium through the Sec pathway. Secretion of the hGH into the medium was verified using SDS-PAGE and western blot analysis. Recombinant hGH was then expressed in E. coli and successfully secreted into cell culture medium via the Sec pathway. The secretion of hGH into the extracellular medium was confirmed by SDS-PAGE and Western blot analysis. Furthermore, the addition of glycine was shown to improve hGH secretion onto the culture medium. Equations for determining the optimal conditions were also determined. Functional hGH analysis using an ELISA-based method confirmed that the ratio of the active form of secreted hGH to the inactive form in the periplasm is higher than this ratio in the cytoplasm.Conclusions: Since the native signal protein peptide of S. aureus protein A was not able to deliver hGH to the extracellular space, it was modified using bioinformatics tools and fused to the n-terminal region of hGh to show that the redesigned signal peptide was functional.

scholarly journals CONSTRUCTION, EXPRESSION AND PURIFICATION OF RECOMBINANT PRE-MATURE PEPTIDE OF PLANTARICIN F FROM Lactobacillus plantarum S34 IN Escherichia coli

2015 ◽  
Vol 16 (1) ◽  
pp. 31 ◽  
Author(s):  
Kusdianawati Kusdianawati ◽  
Apon Zaenal Mustopa ◽  
Suharsono Suharsono ◽  
Bugi Ratno Budiarto ◽  
Fatimah Fatimah ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Lactobacillus Plantarum ◽  
Proteolytic Enzymes ◽  
Leader Peptide ◽  
Human Consumption ◽  
Mature Peptide ◽  
Expression And Purification ◽  
Food Preservative ◽  
E Coli ◽  
Sds Page

Plantaricin is one of bacteriocins that have the potential to be used as food preservative. Plantaricin is safe for human consumption because it can be easily degraded by proteolytic enzymes. The objective of this study was to express and purify recombinant pre-mature peptide of plantaricin F from <em>Lactobacillus plantarum</em> S34 in <em>Escherichia coli</em>. Plantaricin gene-specific primer was used to obtain pln F structural gene amplicon from L. <em>plantarum</em> S34. This amplicon was cloned in pET32a vector and expressed in E. coli BL21 (DE3) pLysS. Pre-mature plantaricin F peptide was expressed as Histagged-fusion protein and separated by Co2+-chelating affinity chromatography. L. <em>plantarum</em> S34-derived pre-mature plantaricin F peptide fused with thioredoxin-(His)6tag had successfully been expressed in E. <em>coli</em> BL21 (DE3) pLysS using pET32a as an expression vector. The fused recombinant pln F as pre-mature state expressed had a molecular mass of +24 kDa, meanwhile the fused recombinant that contained only the leader peptide of pln F appeared as +20 kDa based on SDS-PAGE separations. The optimal production of fused recombinant pln F as soluble fraction was obtained when culture condition was added with 0.5 mM of IPTG and incubated at 22°C for 5 hours (OD~1). Furthermore, the expression of fused recombinant pln F as its pre-mature peptide pointed out that the pln F’s leader peptide could be proteolytically cleaved by a system in heterologous cells. Overall, heterologous pln F production as pre-mature peptide fused with thioredoxin-(His)6tag had been well established. From this research, we expect plantaricin F can be expressed and purified in E. coli.

scholarly journals 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

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.

scholarly journals CLONAGEM, EXPRESSÃO E PURIFICAÇÃO DE UM FRAGMENTO FAB DE ANTICORPO MONOCLONAL MURINO ANTI-PBP2A DE STAPHYLOCOCCUS AUREUS RESISTENTE À METICILINA

2021 ◽  
Author(s):  
Juliana Pascarelli Compan Boechat ◽  
Felipe Rodrigues Semcovici Ramos ◽  
Haroldo Cid Da Silva Junior ◽  
José Procópio Moreno Senna
Keyword(s):  
Staphylococcus Aureus ◽  
Western Blot ◽  
E Coli ◽  
Sds Page

Introdução: Staphylococcus aureus resistente à meticilina (MRSA) é uma das principais bactérias multirresistentes. A resistência se deve principalmente a presença da proteína ligadora de penicilina 2a (PBP2a). Os fragmentos de anticorpos podem ser utilizados de diversas maneiras visando o controle deste patógeno; e a obtenção e purificação eficientes são fundamentais. Objetivo: Realizar a clonagem, expressão e a purificação de fragmentos do tipo Fab murino anti-PBP2a. Metodologia: Foi realizada amplificação das sequências de cadeia leve (CL) e Fd do Fab murino anti-PBP2a por PCR, e ligação das mesmas ao vetor de expressão pCDNA3.4. Após, foi realizada a transformação e clonagem em células de E. coli TOP10, com posterior purificação do material obtido. A orientação e a identidade dos genes clonados no vetor de expressão foram analisadas por sequenciamento nucleotídico. Células HEK293 foram transfectadas com as construções correspondentes a CL e Fd-His, com cultivo acompanhado durante 5 dias. A avaliação da expressão do Fab foi realizada por Western Blot (WB) e SDS-PAGE. O sobrenadante foi coletado para etapa de purificação que foi realizada através de Cromatografia Líquida de Afinidade por Íons Metálicos utilizando a coluna HisTrap™HP. O material alvo de purificação foi injetado à coluna cromatográfica sob fluxo constante de 2mL/min, com o tampão de eluição (20mM fosfato de sódio+0,5M NaCl 1M imizadol, pH 7,4) injetado em gradiente de 0-50% no fluxo de 3mL/min em 30 volumes de coluna. Resultados: Através do sequenciamento dos clones obtidos, determinou-se as construções que apresentavam os genes de CL e Fd na orientação correta para serem utilizados na transfecção. A avaliação da expressão por SDS-PAGE permitiu observar a presença da banda em torno de 50kDa no fim do cultivo (dia 5) e confirmada ser relativa ao Fab murino anti-PBP2a por WB. A purificação do fragmento resultou em uma amostra final com alta homogeneidade, observada em SDS-PAGE. Conclusão: O presente trabalho permitiu a obtenção do Fab com alta homogeneidade, fornecendo a possibilidade da utilização em estudos para avaliação de seu potencial terapêutico e diagnóstico, focando principalmente em inovações diagnósticas como o diagnóstico in situ de focos infecciosos de MRSA.

scholarly journals CLONING, EXPRESSION, AND PARTIAL PURIFICATION OF PLANTARICIN W LOCUS PRODUCED BY Lactobacillus plantarum S34

2017 ◽  
Vol 16 (1) ◽  
Author(s):  
Rifqiyah Nur Umami ◽  
Apon Zaenal Mustopa ◽  
Linda Sukmarini ◽  
Hasim Danuri ◽  
Andini Setyanti Putri ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Western Blot ◽  
Lactobacillus Plantarum ◽  
Partial Purification ◽  
E Coli ◽  
Sds Page

Lactobacillus plantarum S34 dilaporkan mempunyai aktivitas antibakteri yang terkait dengan produksi bakteriosin. Bagian dari gen yang menyandikan salah satu lokus bakteriosin yang diproduksi oleh L. plantarum S34, disebut dengan plantarisin W (plnW), diamplifikasi dari plasmid dan dikloning menggunakan sistem vektor pGEM®-T Easy ke dalam Escherichia coli DH5?. Sekuens nukleotida plnW (± 405 pb) diidentifikasi sebagai protein integral membran. Lebih lanjut, plnW diekspresikan secara heterologus sebagai fusi protein dengan His(6)-tag tioredoksin menggunakan vektor ekspresi pET-32a(+) ke dalam E. coli BL21 (DE3) pLysS. Protein fusi rekombinan plnW terdapat dalam sitoplasma sel, tetapi selain fraksi terlarut terdapat juga fraksi tidak terlarut berupa badan inklusi. Purifikasi parsial dilakukan menggunakan kromatografi afinitas ligan Co2+ untuk fraksi terlarut dan metode elektroelusi gel poliakrilamid untuk fraksi tidak terlarut. Massa molekul berukuran kurang lebih 33 kDa terdeteksi berdasarkan pemisahan SDS-PAGE dan dikonfirmasi dengan Western blot sebagai protein fusi rekombinan plnW. Protein yang sudah terpurifikasi bermanfaat untuk mengetahui kaitan antara struktur dan fungsi bakteriosin.

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