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 EVALUACIÓN BIOQUÍMICA Y BACTERICIDA DEL VENENO DE CROTALUS DURISSUS (DEPARTAMENTO DEL TOLIMA)

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Walter Murillo Arango
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
E Coli ◽  
Sds Page ◽  
Crotalus Durissus

En el departamento del Tolima no hay estudios que permitan precisar con certeza la magnitud del accidente ofídico causado por Crotalus durissus; Por lo anterior existe la necesidad de generar información toxicológica, así como conocimiento del posible potencial antimicrobiano de moléculas como proteínas y péptidos que componen el veneno. En este trabajo se analizó el perfil electroforético por SDS-PAGE del veneno crudo extraído de individuos colectados en el municipio de Natagaima (Tolima) y la asociación de sus componentes con la actividad hemolítica directa e indirecta sobre agar sangre y su actividad bactericida. Se evaluó el potencial del veneno crudo contra Escherichia coli, Staphylococcus aureus y Pseudomonas aeuroginosa. El veneno mostró bandas de peso molecular 26.6 kDa., 17, 14.2, 6.5, 3.5 y 1.06 kDa., correspondientes para reportes del veneno de esta especie. La actividad hemolítica directa e indirecta fueron dosis dependiente, siendo la dosis hemolítica mínima de 200 mg/ml. El efecto bactericida del veneno crudo fue diferencial sobre los microorganismos evaluados, presentando actividad moderada sobre E. coli. Los resultados constituyen datos valiosos para reconocer preliminarmente el potencial antimicrobiano del veneno de Crotalus durissus (cascabel) de la zona de Natagaima-Tolima.

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 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 Changes in protein patterns of Staphylococcus aureus and Escherichia coli by silver nanoparticles capped with poly (4-styrenesulfonic acid-co-maleic acid) polymer

2019 ◽  
Vol 13 (2) ◽  
pp. 39-47 ◽  
Author(s):  
Hathaichanok Tamiyakul ◽  
Sittiruk Roytrakul ◽  
Janthima Jaresitthikunchai ◽  
Narumon Phaonakrop ◽  
Somboon Tanasupawat ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Silver Nanoparticles ◽  
Maleic Acid ◽  
Molecular Transport ◽  
Protein Translation ◽  
Bacterial Protein ◽  
Protein Patterns ◽  
E Coli ◽  
Sds Page

Abstract Background While silver nanoparticles (AgNPs) are increasingly attractive as an antibacterial agent in many applications, the effect of AgNPs on bacterial protein profiles, especially AgNPs stabilized by polymeric molecules, is not well understood. Objectives To investigate the changes in bacterial protein patterns by AgNPs capped with poly (4-styrenesulfonic acid-co-maleic acid) (AgNPs-PSSMA) polymer toward Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922. Methods The growth of bacteria after incubated with AgNPs-PSSMA for different time intervals was determined by optical density at 600 nm. Their protein patterns were observed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the proteomic analysis of extracted proteins was determined by liquid chromatography-tandem mass spectrometry (LC–MS/MS). Results AgNPs-PSSMA was able to inhibit the growth of both S. aureus and E. coli cells. The treated bacterial cells expressed more proteins than the untreated cells as seen from SDS-PAGE study. Nanosilver (NS) caused the upregulation of metabolic gene, waaA, in S. aureus cells. For E. coli cells, the upregulated proteins were metabolic genes (srlB, fliE, murD) and other genes dealt with DNA replication (dinG), DNA–RNA transcription (yrdD), RNA– protein translation (rplD), molecular transport (sapF), and signal transduction (tdcF). Conclusions The antibacterial effect of AgNPs-PSSMA may arise by changing the bacterial proteins and thus interfering with the normal cell function.

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.

scholarly journals Novel Cytoplasmic Bacteriocin Compounds Derived from Staphylococcus epidermidis Selectively Kill Staphylococcus aureus, Including Methicillin-Resistant Staphylococcus aureus (MRSA)

Pathogens ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 87 ◽  
Author(s):  
In-Taek Jang ◽  
Miso Yang ◽  
Hwa-Jung Kim ◽  
Jeong-Kyu Park
Keyword(s):  
Staphylococcus Aureus ◽  
Staphylococcus Epidermidis ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Methicillin Resistance ◽  
Precipitation Method ◽  
E Coli ◽  
Sds Page ◽  
Zones Of Inhibition ◽  
Agar Well Diffusion Assay ◽  
Diffusion Assay

Staphylococcus aureus (S. aureus) is one of the well-known agents causing atopic dermatitis (AD) in susceptible individuals, and Staphylococcus epidermidis (S. epidermidis) produces class I thermostable bacteriocins that can selectively kill S. aureus, suggesting protective roles against AD. There is a large need for developing precise therapies only to target S. aureus and not to harm the beneficial microbiome. On the agar well diffusion assay, live planktonic S. epidermidis showed clear zones of inhibition of S. aureus growth, but heat-killed cells and cell-free supernatants did not show this. These results would lead us to hypothesize that cytoplasmic bacteriocin from S. epidermidis will be a promising agent to inhibit S. aureus growth. Therefore, we have extracted a novel thermolabile cytoplasmic bacteriocin from S. epidermidis using trichloroactic acid (TCA)/acetone precipitation method after cell lysis with a SDS-containing buffer. These bacteriocin selectively exhibited antimicrobial activity against S. aureus and methicillin-resistance Staphylococcus aureus (MRSA), presenting no active actions against S. epidermidis, E. coli, and Salmonella Typhimurium. The extracted cytoplasmic bacteriocin compounds revealed several diffuse bands of approximately 40–70 kDa by SDS-PAGE. These findings suggest that these cytoplasmic bacteriocin compounds would be a great potential means for S. aureus growth inhibition and topical AD treatment.

scholarly journals NGHIÊN CỨU BIỂU HIỆN GEN CRY2A DIỆT ẤU TRÙNG RUỒI NHÀ (MUSCA DOMESTICA) CỦA CHỦNG BACILLUS THURINGIENSIS SEROVAR KURSTAKI MSS8.4

2018 ◽  
Vol 15 (3) ◽  
pp. 563-569
Author(s):  
Phạm Thùy Dương ◽  
Ngô Đình Bính ◽  
Trịnh Thị Thu Hà ◽  
Lê Đức Khánh
Keyword(s):  
Amino Acid ◽  
Bacillus Thuringiensis ◽  
Western Blot ◽  
Musca Domestica ◽  
E Coli ◽  
Sds Page

Các độc tố Cry2A do gen cry2A mã hóa là đặc biệt quan trọng vì chúng có hoạt tính diệt nhiều loại côn trùng khác nhau bao gồm: côn trùng bộ Cánh vảy và côn trùng bộ Hai cánh. Mục tiêu của nghiên cứu này nhằm biểu hiện protein Cry2A tái tổ hợp trong E. coli, là cơ sở để nghiên cứu tạo ra chế phẩm sinh học diệt côn trùng bộ hai cánhtừ vi khuẩn Bacillus thuringiensis. B. thuringiensis serovar kurstaki MSS8.4 là chủng được phân lập từ đất thuộc huyện Sóc Sơn – TP Hà Nội. Chủng MSS8.4 có khả năng sinh tổng hợp đồng thời 2 loại tinh thể hình lưỡng tháp và hình khối lập phương, có khả năng diệt ấu trùng thuộc bộ Cánh vẩy và bộ Hai cánh. Đoạn gen mã hóa cho protein Cry2A có kích thước khoảng 1,9 kb được khuếch đại bởi cặp mồi đặc hiệu. Trình tự gen cry2A của chủng MSS8.4 được đăng k. trên ngân hàng gen quốc tế với mã số: KM588296 có độ tương đồng 99% và có 6 vị trí sai khác so với trình tự gen cry2A trên ngân hàng gen. Các vị trí đó bao gồm: 305 (G-A), 500 (G-A), 783 (T-G), 1054 (T-G), 1303 (G-A), 1575 (C-T). Tuy nhiên, trình tự amino acid của protein cry2A suy diễn có độ tương đồng 99% và chỉ có 5 vị trí sai khác (102 (S-N), 167 (R-Q), 261 (F-L), 352 (W-G), 435 (D-N)), đột biến nucleotide ở vị trí 1575 (C-T) không làm thay thế amino acid. Tiếp đó, trình tự gen này đã được đưa vào vector pET-22b(+) và biểu hiện trong tế bào E. coli BL21 (DE3) ở dạng dung hợp với đoạn pelB ở đầu N và đuôi ái lực (His)6 ở đầu C. Các kết quả điện di protein SDS-PAGE và Western blot cho thấy protein tái tổ hợp Cry2A đã được tạo ra thành công với hiệu suất lớn trong tế bào E. coli. Kết quả thử nghiệm với ruồi nhà M. domestica cho thấy rCry2A có hoạt tính cao với côn trùng thử nghiệm (LC50 = 264,7 μg).

The porcine glucocorticoid receptor: sequencing a 2.1 kb cDNA fragment and raising specific polyclonal antibodies for western blotting, immunoprecipitation and immunohistochemistry

2001 ◽  
Vol 73 (2) ◽  
pp. 267-278 ◽  
Author(s):  
M. Gutscher ◽  
S. Eder ◽  
M. Müller ◽  
R. Claus
Keyword(s):  
Glucocorticoid Receptor ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Polyclonal Antibodies ◽  
Cdna Fragment ◽  
E Coli ◽  
Denatured Protein ◽  
Sds Page ◽  
Receptor Fragment

AbstractGlucocorticoids and their tissue receptors are involved in many metabolic and developmental processes. Until now only two short fragments with a total length of 200 amino acids were known from the glucocorticoid receptor of the pig. Therefore we sequenced the main part (2.1 kb) of the porcine receptor. In addition, we subcloned a cDNA fragment of this sequence coding for 135 aa of the modulatory region in a pET expression vector. The protein fragment was expressed in E. coli as a his-tag fusion protein. In the SDS-PAGE, the crude E. coli extracts showed an enrichment of a 15 kDa protein which corresponds to the estimated molecular weight for the receptor fragment.After lysis and Ni-NTA affinity chromatography under denaturing conditions the protein was further purified either by dialysis (native protein) or by SDS-PAGE (linearized form). Both forms were emulsified together in adjuvant and used for rabbit immunization.The resulting antibodies were characterized by western blot analysis, immunoprecipitation, and additionally by immunohistochemistry. Western blot analysis confirmed the binding of the denatured protein by the antiserum and revealed a high binding affinity. Immunoprecipitation demonstrated that both the occupied and unoccupied forms of the receptor are detected. The specificity of the antiserum for pGCR was additionally demonstrated by immunohistochemistry.

scholarly journals Produção de antissoro policlonal utilizando a proteína capsidial recombinante do Rupestris stem pitting-associated virus

2010 ◽  
Vol 40 (11) ◽  
pp. 2385-2388 ◽  
Author(s):  
Marcos Fernando Basso ◽  
Thor Vinícius Martins Fajardo ◽  
Marcelo Eiras ◽  
Ricardo Antônio Ayub ◽  
Osmar Nickel
Keyword(s):  
Escherichia Coli ◽  
Western Blot ◽  
E Coli ◽  
Sds Page ◽  
Rupestris Stem Pitting ◽  
Stem Pitting

O Rupestris stem pitting-associated virus (RSPaV) é o agente causal das caneluras do lenho da videira. Este trabalho teve como objetivo produzir antissoro policlonal a partir da proteína capsidial (CP) recombinante do RSPaV e avaliar a sua especificidade e sensibilidade. O gene da CP do RSPaV, com 780pb, foi previamente caracterizado. Esse gene foi subclonado no sítio de restrição EcoRI, no vetor de expressão pRSET-B e o plasmídeo recombinante foi utilizado para induzir a expressão da CP em Escherichia coli. A CP, ligada a uma cauda de seis histidinas, foi purificada por meio de cromatografia de afinidade em coluna de Ni-NTA a partir do extrato de proteínas totais extraídas de E. coli. A identidade da proteína purificada foi confirmada em SDS-PAGE e Western blot, utilizando-se anticorpos comerciais contra a cauda de seis histidinas. A CP recombinante expressada in vitro apresentou massa molecular de cerca de 31kDa. A proteína purificada foi quantificada e 2,55mg foram utilizados para a imunização de um coelho. O antissoro policlonal obtido reagiu com diferentes isolados deste vírus, extraídos de videiras em ELISA indireto.

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.

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