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

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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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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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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Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product

Biochemical Journal ◽

10.1042/bj2480043 ◽

1987 ◽

Vol 248 (1) ◽

pp. 43-51 ◽

Cited By ~ 17

Author(s):

J Charlier ◽

R Sanchez

Keyword(s):

Escherichia Coli ◽

Gene Product ◽

Activity Ratio ◽

Deae Cellulose ◽

Trna Synthetase ◽

Trna Synthetases ◽

E Coli ◽

Aminoacyl Trna Synthetases

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5′)tetraphospho(5′)adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.

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Cloning and Over-expression of xynB Gene of Bacillus subtilis subsp. spizizenii W23 into Escherichia coli Origami Host Cells

KnE Life Sciences ◽

10.18502/kls.v3i5.986 ◽

2017 ◽

Vol 3 (5) ◽

pp. 139

Author(s):

Mariana Wahjudi ◽

Catherina . ◽

Nita Marcelia Wangunhardjo ◽

Ernest Suryadjaja ◽

Xavier Daniel

Keyword(s):

Escherichia Coli ◽

Bacillus Subtilis ◽

Recombinant Plasmid ◽

Sodium Dodecyl ◽

Xylanase Activity ◽

Cellular Protein ◽

Host Cells ◽

Chromosomal Dna ◽

Clear Zone ◽

Sds Page

The xynB gene of Bacillus subtilis subsp. spizizenii W23 is predicted to encode a xylan 1,4-beta-xylosidase. Application of XynB enzymes in industries is wide. Production of this enzyme in its host cells is naturally restricted by repression process. It will give certain beneficial to over-expressed the enzymes in other host-cells under inducing promoter. This study aimed to clone the xynB gene from Bacillus subtilis subsp. spizizenii W23, to pMMB67EH plasmid, and to over-express the xynB gene in Escherichia coli Origami as host cells. The xynB gene was successfully amplified by polymerase chain reaction (PCR) technique using a pair of primers flanking the gene sequence and chromosomal DNA of the W23 strain as a template. The xynB gene inserted in recombinant plasmid was confirmed by PCR detection using primers pair’s specific for xynB gene and for the vector, then continued by restriction analyses. The result showed that transformants clone 9 and 10 bear the recombinant pMMB-xynB plasmid. The xylanase activity of xynB gene in Escherichia coli Origami clone 10 was detected by sodium-dodecyl-sulfate polyacrylamide gel analyses and with addition of isopropyl-β-D-thio-galactoside (IPTG) as an inducer. The protein seem to be over-expressed as intra- and extra-cellular protein detected on SDS-PAGE gel. Result from xylan degrading activity on Luria-Bertani-xylan-IPTG plate with addition of Congo Red, showed that the cells with pMMB-xynB recombinant plasmid have clear zone around the colonies while the transformant bearing an empty plasmid showed no clear zone. It could be concluded that the xynB gene of Bacillus subtilis subsp.spizizenii W23 has been successfully been cloned on pMMB67EH plasmid and over-expressed in the Escherichia coli Origami cells as intra- and extra-cellular protein, as observed on SDS-PAGE gel analysis. The protein has activity on xylan degradation.

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Evidence for the involvement of a 66 kDa membrane protein in the synthesis of sterolglucoside in Saccharomyces cerevisiae.

Acta Biochimica Polonica ◽

10.18388/abp.1995_4657 ◽

1995 ◽

Vol 42 (2) ◽

pp. 269-274 ◽

Cited By ~ 2

Author(s):

U Lenart ◽

J Haplova ◽

P Magdolen ◽

V Farkas ◽

G Palamarczyk

Keyword(s):

Saccharomyces Cerevisiae ◽

Molecular Mass ◽

Deae Cellulose ◽

Yeast Saccharomyces Cerevisiae ◽

Sds Page ◽

Nonionic Detergent ◽

Membrane Bound ◽

Labeled Probe ◽

Triton X ◽

Cellulose Column

The membrane-bound sterolglucoside synthase from the yeast Saccharomyces cerevisiae has been solubilized by nonionic detergent, Nonidet P-40, Triton X-100, and partially purified by DEAE-cellulose column chromatography and ammonium sulfate fractionation. SDS/PAGE of the purified fraction revealed the presence of two protein bands of molecular mass 66 kDa and 54 kDa. In an attempt to identify further the polypeptide chain of sterolglucoside synthase, the partially purified enzyme was treated with [di-125I]-5-[3-(p-azidosalicylamide)]allyl-UDPglucose, a photoactive analogue of UDP glucose, which is a substrate for this enzyme. Upon photolysis the 125I-labeled probe was shown to link covalently to the 66 kDa protein. The photoinsertion was competed out by the presence of unlabeled UDPglucose thus suggesting that this protein contains substrate binding site for UDPglucose. Since photoinsertion of the probe to protein of 66 kDa correlates with the molecular mass of the protein visualized upon enzyme purification we postulate that the 66 kDa protein is involved in sterolglucoside synthesis in yeast.

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

Indonesian Journal of Agricultural Science ◽

10.21082/ijas.v16n1.2015.31-38 ◽

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 Lactobacillus plantarum S34 in Escherichia coli. Plantaricin gene-specific primer was used to obtain pln F structural gene amplicon from L. plantarum 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. plantarum S34-derived pre-mature plantaricin F peptide fused with thioredoxin-(His)6tag had successfully been expressed in E. coli 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.

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

Avicenna Journal of Medical Biotechnology ◽

10.18502/ajmb.v13i4.7204 ◽

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).

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

Blood ◽

10.1182/blood.v73.5.1202.1202 ◽

1989 ◽

Vol 73 (5) ◽

pp. 1202-1206 ◽

Cited By ~ 7

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.

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

Revista do Instituto de Medicina Tropical de São Paulo ◽

10.1590/s0036-46651996000600003 ◽

1996 ◽

Vol 38 (6) ◽

pp. 401-406 ◽

Cited By ~ 3

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

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Purification and characterization of two fructose diphosphate aldolases from Escherichia coli (Crookes' strain)

Biochemical Journal ◽

10.1042/bj1310833 ◽

1973 ◽

Vol 131 (4) ◽

pp. 833-841 ◽

Cited By ~ 67

Author(s):

Donald Stribling ◽

Richard N. Perham

Keyword(s):

Escherichia Coli ◽

Molecular Weight ◽

Metal Ions ◽

Gel Filtration ◽

Deae Cellulose ◽

Class Ii ◽

Class I ◽

E Coli ◽

Km Value ◽

Bivalent Metal Ions

Two fructose diphosphate aldolases (EC 4.1.2.13) were detected in extracts of Escherichia coli (Crookes' strain) grown on pyruvate or lactate. The two enzymes can be resolved by chromatography on DEAE-cellulose at pH7.5, or by gel filtration on Sephadex G-200, and both have been obtained in a pure state. One is a typical bacterial aldolase (class II) in that it is strongly inhibited by metal-chelating agents and is reactivated by bivalent metal ions, e.g. Ca2+, Zn2+. It is a dimer with a molecular weight of approx. 70000, and the Km value for fructose diphosphate is about 0.85mm. The other aldolase is not dependent on metal ions for its activity, but is inhibited by reduction with NaBH4 in the presence of substrate. The Km value for fructose diphosphate is about 20μm (although the Lineweaver–Burk plot is not linear) and the enzyme is probably a tetramer with molecular weight approx. 140000. It has been crystallized. On the basis of these properties it is tentatively assigned to class I. The appearance of a class I aldolase in bacteria was unexpected, and its synthesis in E. coli is apparently favoured by conditions of gluconeogenesis. Only aldolase of class II was found in E. coli that had been grown on glucose. The significance of these results for the evolution of fructose diphosphate aldolases is briefly discussed.

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