A novel protein refolding protocol for the solubilization and purification of recombinant peptidoglycan-associated lipoprotein from Xylella fastidiosa overexpressed in Escherichia coli

Santos, Clelton A.; Beloti, Lilian L.; Toledo, Marcelo Augusto Szymanski de; Crucello, Aline; Favaro, Marianna T.P.; Mendes, Juliano S.; Santiago, André S.; Azzoni, Adriano Rodrigues; Souza, Anete Pereira de

doi:10.1016/j.pep.2012.01.010

Cited by 21 publications

(17 citation statements)

References 28 publications

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“…[16] Also, the dialysis of the protein of interest in two times, as described above in the presence of glycerol and a concentration of EDTA that let any denatured protein -if present-effected by urea to renature (refolded) and retain its biological normal form again. [15] Ion-exchange is a largely used system for separating biomolecules according to differences in ionic charge, in this study we used primarily a weak anion exchange column, the DEAE Sepharose column for terminating the action of Enterokinase enzyme, by separating the Parathyroid hormone (target protein) after proteolysis of fusion protein by Enterokinase from the non-proteolysis fusion protein and the fusion partner (TrxA-His. Tag) of the proteolysis protein as well as all the proteolysis reaction products, this was carried out depending on proteins PI and the surrounding PH.…”

Section: Discussionmentioning

confidence: 99%

“…The binding and washing buffer used for HisLink TM Protein Purification consisted of 50 mM phosphate buffer (NaH 2 PO 4 ) PH = 8, 300 mM NaCl, 5 mM Imidazole and Up to 8 M Urea, while, the elution buffer contained the same component like binding and washing buffer in addition to 250 mM Imidazole. Then, the product of the HisLink TM Protein Purification system was dialyzed using dialysis tube as described by Santos et al [15] The recombinant protein was dialyzed in a buffer consisted of 50 mM phosphate buffer (NaH 2 PO 4 ), 100 mM NaCl, 0.1 Mm EDTA and 10% v/v glycerol (PH = 8). The volume of the dialysis buffer was primarily equal to 10X of sample volume (10 ml of sample was dialyzed in a 100 ml of dialysate).…”

Section: Cell Lysate Preparation and Expression Analysismentioning

confidence: 99%

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Extraction and purification of recombinant intact human Parathyroid Hormone (hPTH) from bacterial cell

Al-Badran

Abdul-Jabbar

2017

JBEI

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Objective: The intact human Parathyroid hormone (hPTH) is one of the biopharmaceutical drug produced by biotechnology, this hormone can be provided in a good amount using simple batch culture, and therefore the main purpose of this study was the production of purified bioactive intact hPTH. Methods: A cloning BL21(DE3) strain (which already cloned in our Lab. by synthetic human Parathyroid hormone (shPTH gene) was grown in LB medium and the cloning gene expression was induced by addition of IPTG to the medium. The recombinant fused protein was purified from the bacterial cell lysate by affinity chromatography and underwent proteolysis by Enterokinase enzyme to obtain the target hPTH, and it's further purified by DEAE and SP Sepharose ion exchange chromatography. RP-HPLC analysis and biological activity on the MCF-7 breast cancer cells were done for both the standard and produced hPTH. Furthermore, the haemolysis ability of the latter was tested on the human RBC. Results: 225 ng/ml of hPTH was produced after SP chromatography which detected by specific PTH ELISA kit, standard and produced hPTH showed the same peaks in the same retention time when analyzed by HPLC. The produced hPTH haemolysis assay showed the ability of the produced hPTH to partially haemolyze human RBC in a concentration above 200 µg/ml. The bioactivity assay for the produced and standard rhPTH demonstrated that both compounds had a biological activity on the MCF-7 cells with IC50 of about 84.4 and 75.7 µg/ml for standard and produced hPTH respectively, and no significant difference was detected between them. Conclusions: Via suitable purification processes, the biologically active hPTH with structure similar to the standard ones as detected by RP-HPLC and bioactivity assay, can be obtained by using already cloned isolate with shPTH gene for hormone production. The hormone showed haemolysis effect on the RBC similar to the normal secreted hPTH.

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Section: Discussionmentioning

confidence: 99%

Section: Cell Lysate Preparation and Expression Analysismentioning

confidence: 99%

Extraction and purification of recombinant intact human Parathyroid Hormone (hPTH) from bacterial cell

Al-Badran

Abdul-Jabbar

2017

JBEI

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“…The dynamics of inclusion body formation are such that there are typically a small number of inclusion bodies seeded by an incorrectly folded protein intermediate. Although various studies have suggested refolding strategies both in vivo (Zhao et al, 2012) and in vitro (Santos et al, 2012), the consensus is that it is critical to prevent inclusion body formation from ever occurring to maximize yield (Schein, 1989; Fink, 1998). …”

Section: Commentarymentioning

confidence: 99%

Characterizing Protein Kinase Substrate Specificity Using the Proteomic Peptide Library (ProPeL) Approach

Lubner

Balsbaugh

Church

et al. 2018

CP Chemical Biology

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Characterizing protein kinase substrate specificity motifs represents a powerful step in elucidating kinase-signaling cascades. The protocol described here uses a bacterial system to evaluate kinase specificity motifs in vivo, without the need for radioactive ATP. The human kinase of interest is cloned into a heterologous bacterial expression vector and allowed to phosphorylate E. coli proteins in vivo, consistent with its endogenous substrate preferences. The cells are lysed, and the bacterial proteins are digested into peptides and phosphoenriched using bulk TiO . The pooled phosphopeptides are identified by tandem mass spectrometry, and bioinformatically analyzed using the pLogo visualization tool. The ProPeL approach allows for detailed characterization of wildtype kinase specificity motifs, identification of specificity drift due to kinase mutations, and evaluation of kinase residue structure-function relationships. © 2018 by John Wiley & Sons, Inc.

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“…• C and with 100X from the same solution for 16h at 4 • C. [12] Treatment with Factor Xa: To remove His-tag from produced protein, factor Xa (Biolab, U.K.) was used by incubation for 6 h at 23…”

Section: Hs Gene Expression In Bl21(de3)mentioning

confidence: 99%

Production and purification of constructed recombinant hirudin in BL21(DE3) strain

Al-Badran

Al-Fadal

2017

JBEI

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Background/Objective: Hirudin, an extract from the leech, has powerful antithrombin activity affecting the blood coagulation pathway, it is the most potent natural inhibitor of thrombin, it binds thrombin with high affinity, so, the aim of this study was to build hirudin gene by overlapping extension PCR then cloning and expression in BL21(DE3) strain. Methods: Hirudin gene constructed with four modified primers then the final product amplified by two primers named as A, B by using overlapping extension PCR, for gene expression, BL21(DE3) strain was used under the control of T7 promoter in pET-16b vector and for hirudin production, LB broth medium was used as fermentation medium, to detect the expression of hirudin in BL21(DE3) strain in fermentation media real-time PCR was used. Hirudin protein purified at first by (Immobilized metal affinity chromatography) IMAC, then this protein was dialysis and treated with factor Xa to eliminate His-tag. Then hirudin purified by DEAE sepharose and SP sepharose column, the concentration of protein determined by ELISA, furthermore the activity evaluated by thrombin titration and activated partial thromboplastin time (APPT) test. Results: Hirudin gene constructed in two round PCR first round produced two products (product 1,117 bp while product 2,114 bp) the second-round PCR gave the final product 213 bp. The resulted band from gel electrophoresis for constructed vector pET-16b-HirudinS was (5,901 bp). Hirudin expression was established by real-time PCR with Ct value (20.28). The analysis on 15% SDS-PAGE for the SP sepharose column illustrated the hirudinS protein band with size about ∼10.8. Concentration of produced hirudin within its solution reached to 1.75 ng, thrombin titration method showed that the hirudin protein required 300 µl from thrombin to clot, also, APPT test showed that hirudin elongated clotting time to 7 min in comparison with 6min for aspirin and the statistical analysis results for APPT test illustrated that there was no significant difference between hirudin and aspirin. Conclusion: This study approved that overlapping extension PCR is a good strategy for building hirudin gene and it's successfully expressed in BL21(DE3) strain.

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A novel protein refolding protocol for the solubilization and purification of recombinant peptidoglycan-associated lipoprotein from Xylella fastidiosa overexpressed in Escherichia coli

Cited by 21 publications

References 28 publications

Extraction and purification of recombinant intact human Parathyroid Hormone (hPTH) from bacterial cell

Extraction and purification of recombinant intact human Parathyroid Hormone (hPTH) from bacterial cell

Characterizing Protein Kinase Substrate Specificity Using the Proteomic Peptide Library (ProPeL) Approach

Production and purification of constructed recombinant hirudin in BL21(DE3) strain

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