scholarly journals Estudo da superexpressão da enzima B-galactosidase recombinante de kluyveromyces sp. Em diferentes Cepas de escherichia coli

ScientiaTec ◽  
2020 ◽  
Vol 7 (03) ◽  
Author(s):  
Bruna Coelho de Andrade ◽  
Victória Furtado Migliavacca ◽  
Vanessa Yuki Grafulin ◽  
Claucia Fernanda Volken de Souza ◽  
Jocelei Maria Chies ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
E Coli ◽  
Sds Page

A enzima B-galactosidase hidrolisa a lactose em glicose e galactose. É utilizada para produção de alimentos com baixo teor deste açúcar. A obtenção dessa enzima recombinante poderá permitir sua produção em maiores quantidades com um menor custo. O objetivo deste trabalho foi estudar a superexpressão da enzima B-galactosidase recombinante de Kluyveromyces sp. em diferentes cepas de E. coli. O gene da B-galactosidase foi amplificado e ligado a um vetor de clonagem e subclonado em vetor de expressão. Para os testes de expressão, foram testadas diferentes cepas eletrocompetentes de E. coli: BL21 (DE3), Rosetta (DE3), Rosetta-gami 2 (DE3), C41 (DE3) e C43 (DE3); diferentes meios de cultura: LB, TB e M9; temperaturas de cultivo: 30°C e 37°C; e concentrações do indutor isopropil-tiogalactosídeo (IPTG): 0,05 mM e 1 mM. Foram realizadas coletas nos tempos 3 h, 6 h, 9 h, 24 h e 27 h após a indução. A expressão da enzima foi analisada por SDS-PAGE, quantificada pelo método de Bradford e sua atividade enzimática foi determinada usando o-nitrofenol-B-D-galactopiranoside (ONPG) como substrato. A cepa BL21 (DE3) obteve a maior atividade enzimática específica, aproximadamente 250 U/mgproteína, em LB, à 30°C, após 9 horas de indução com 0,05 mM de IPTG.

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.

Production of PEGylated GCSF from Non-classical Inclusion Bodies Expressed in Escherichia coli

2021 ◽  
Author(s):  
Nguyen Thi My Trinh ◽  
Tran Linh Thuoc ◽  
Dang Thi Phuong Thao
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Insoluble Fraction ◽  
Exchange Chromatography ◽  
Native Page ◽  
E Coli ◽  
Sds Page ◽  
Stimulating Factor

Background: The recombinant human granulocyte colony stimulating factor con-jugated with polyethylene glycol (PEGylated GCSF) has currently been used as an efficient drug for the treatment of neutropenia caused by chemotherapy due to its long circulating half-life. Previous studies showed that Granulocyte Colony Stimula-ting Factor (GCSF) could be expressed as non-classical Inclusion Bodies (ncIBs), which contained likely correctly folded GCSF inside at low temperature. Therefore, in this study, a simple process was developed to produce PEGylated GCSF from ncIBs. Methods: BL21 (DE3)/pET-GCSF cells were cultured in the LiFlus GX 1.5 L bioreactor and the expression of GCSF was induced by adding 0.5 mM IPTG. After 24 hr of fermentation, cells were collected, resuspended, and disrupted. The insoluble fraction was obtained from cell lysates and dissolved in 0.1% N-lauroylsarcosine solution. The presence and structure of dissolved GCSF were verified using SDS-PAGE, Native-PAGE, and RP-HPLC analyses. The dissolved GCSF was directly used for the con-jugation with 5 kDa PEG. The PEGylated GCSF was purified using two purification steps, including anion exchange chromatography and gel filtration chromatography. Results: PEGylated GCSF was obtained with high purity (~97%) and was finally demonstrated as a form containing one GCSF molecule and one 5 kDa PEG molecule (monoPEG-GCSF). Conclusion: These results clearly indicate that the process developed in this study might be a potential and practical approach to produce PEGylated GCSF from ncIBs expressed in Escherichia coli (E. coli).

scholarly journals Expression in Escherichia coli of the human fibrinogen B beta chain and its cleavage by thrombin

Blood ◽  
1989 ◽  
Vol 73 (5) ◽  
pp. 1202-1206 ◽  
Author(s):  
MG Bolyard ◽  
ST Lord
Keyword(s):  
Escherichia Coli ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Dependent Manner ◽  
Beta Chain ◽  
Gamma Chain ◽  
Human Fibrinogen ◽  
E Coli ◽  
Sds Page

Abstract The human fibrinogen B beta chain was expressed in Escherichia coli to study the functions of fibrinogen associated with this subunit. Recombinant B beta chains were expressed at 100 ng/mL in an IPTG- dependent manner. A first cistron sequence, inserted into the expression vector 5′ to the B beta chain cDNA, was required to express the protein. Recombinant B beta chains were expressed within five minutes after induction with IPTG and were soluble in physiologic buffers. The recombinant B beta chains migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at a rate identical to B beta chains from fibrinogen treated with N-glycanase. Recombinant B beta chains were cleaved by thrombin, as demonstrated by the loss of cross-reactivity with a monoclonal antibody (MoAb) specific for the undigested B beta 1–42 fragment. The levels of expression of the B beta chain were much lower than those reported previously for the gamma chain of fibrinogen expressed in a similar vector in E coli. However, these levels are sufficient to allow further characterization of this fibrinogen subunit.

scholarly journals Purification and parcial characterization of a hemagglutinating factor (HAF): a possible adhesive factor of the diffuse adherent of Escherichia coli (DAEC)

1996 ◽  
Vol 38 (6) ◽  
pp. 401-406 ◽  
Author(s):  
Yano Tomomasa ◽  
Cleide Ferreira Catani ◽  
Michiko Arita ◽  
Takeshi Honda ◽  
Toshio Miwatani
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Hela Cells ◽  
Immunofluorescence Test ◽  
Native Form ◽  
Bacterial Surface ◽  
E Coli ◽  
Sds Page ◽  
Adhesive Factor

The mannose-resistant hemagglutinating factor (HAF) was extracted and purified from a diffuse adherent Escherichia coli (DAEC) strain belonging to the classic enteropathogenic E. coli (EPEC) serotype (0128). The molecular weight of HAF was estimated to be 18 KDa by SDS-PAGE and 66 KDa by Sephadex G100, suggesting that the native form of HAF consists of 3-4 monomeric HAF. Gold immunolabeling with specific HAF antiserum revealed that the HAF is not a rigid structure like fimbriae on the bacterial surface. The immunofluorescence test using purified HAF on HeLa cells, in addition to the fact that the HAF is distributed among serotypes of EPEC, suggests that HAF is a possible adhesive factor of DAEC strains

A fraction from Escherichia coli with anti-Aspergillus properties

2005 ◽  
Vol 54 (4) ◽  
pp. 375-379 ◽  
Author(s):  
V Yadav ◽  
R Mandhan ◽  
Rajesh Dabur ◽  
A K Chhillar ◽  
J Gupta ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Concentration ◽  
Exchange Chromatography ◽  
Active Molecule ◽  
Standard Drug ◽  
E Coli ◽  
Variable Activity ◽  
Sds Page ◽  
Haemolytic Assay

The products of various strains of Escherichia coli (BL21, DH5α, HB101 and XL Blue) were investigated for antimycotic properties using pathogenic isolates of Aspergillus. Co-culture experiments revealed that E. coli strains exhibited variable activity against Aspergillus fumigatus. The lysates prepared from DH5α, HB101 and XL Blue strains of E. coli showed inhibitory activity against A. fumigatus in the protein concentration range of 62.50 to 250.00 μg ml−1. The highest activity was seen in the lysate of BL21, which inhibited the growth of A. fumigatus and Aspergillus flavus completely at a concentration of 31.25 μg protein ml−1. The MIC of BL21 lysate against Aspergillus niger was found to be 62.50 μg ml−1. The in vitro toxicity of BL21 lysate was evaluated using a haemolytic assay. A BL21 lysate protein concentration of 1250.00 μg ml−1 was found to be nontoxic to human erythrocytes. The standard drug amphotericin B lysed 100 % of erythrocytes at a concentration of 37.50 μg ml−1. SDS-PAGE showed the presence of at least 15 major proteins in the lysate of BL21. Ion-exchange chromatography resolved the BL21 lysate into five fractions and fraction III was found to be endowed with anti-Aspergillus properties. The MIC of this fraction was found to be 3.90 μg ml−1. Further work on the purification of the active molecule and its characterization is in progress.

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.

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 Escherichia coli using modified staphylococcal protein a signal peptide

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Garshasb Rigi ◽  
Amin Rostami ◽  
Habib Ghomi ◽  
Gholamreza Ahmadian ◽  
Vasiqe Sadat Mirbagheri ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Hormone ◽  
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 Escherichia 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 TẠO DÒNG VÀ BIỂU HIỆN GENE MÃ HÓA KHÁNG NGUYÊN RHOPTRY-ASSOCIATED PROTEIN-1 CỦA BABESIA BOVIS TRONG ESCHERICHIA COLIBL21 (DE3)

2020 ◽  
Vol 129 (3A) ◽  
Author(s):  
Đinh Thị Bích Lân ◽  
Lê Đức Thạo ◽  
Lê Quốc Việt ◽  
Đặng Thị Hương ◽  
Lê Viết Quân ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Babesia Bovis ◽  
E Coli ◽  
Escherichia Coli Bl21 ◽  
Sds Page

Tóm tắt: Trong nghiên cứu này chúng tôi đã tạo dòng và biểu hiện thành công đoạn gene mã hóa kháng nguyên Rhoptry-Associated Protein (RAP-1) của Babesia bovis phân lập từ mẫu máu bò thu thập tại tỉnh Thừa Thiên Huế. Đoạn gene mã hóa cho kháng nguyên RAP-1 được tạo dòng và gắn vào plasmid pGEX-4T-1, sau đó biến nạp vào chủng Escherichia coli BL21 (DE3). Kết quả cho thấy đoạn gene mã hóa kháng nguyên RAP-1 có chiều dài 300 bp, mã hóa chuỗi polypeptide dài 100 axit amin, tương đồng 99% so với đoạn gene mã hóa cho RAP-1 đã được công bố trên GenBank (LC157851). Kết quả điện di trên SDS-PAGE cho thấy protein dung hợp GST-RAP-1 có khối lượng phân tử khoảng 38kDa.Từ khóa: Babesia bovis, RAP-1, pGEX-4T-1, E. coli BL21, tạo dòng, Thừa Thiên Huế

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

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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