scholarly journals Production of the 13C/15N single-labeled insecticidal protein Cry1Ab/Ac for the assessment of metabolic fate using recombinant Escherichia coli

2020 ◽  
Author(s):  
Zibo Wang ◽  
Cong Hu ◽  
Yu Sun ◽  
Wei Jiang ◽  
Guogan Wu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Stable Isotope ◽  
Inclusion Bodies ◽  
Isotope Labeling ◽  
Recombinant Expression ◽  
Insecticidal Protein ◽  
Cry Protein ◽  
Environmental Behaviors ◽  
Cry Proteins ◽  
Sds Page

Abstract Stable isotope-labeled Cry1Ab/Ac protein is necessary for the metabolic study of exogenous insecticidal protein in soil using the stable isotope labeling technique, but no recombinant expression protocols for this protein have been reported. The artificially synthesized gene Cry1Ab/Ac of Bt rice Huahui No. 1, which obtained the safety certificate in China, was subcloned into pUC57 in this study, and the expression vector pET-28a-CryAb/Ac was constructed and transformed into Escherichia coli BL21 (DE3) competent cells. Next, 0.2 mM IPTG was added to these cells and cultured at 37°C for 4 h to induce the expressed protein to form inclusion bodies in the presence of M9 medium containing either [U-13C] glucose (5% 13C-enriched) or [15N] ammonium chloride (5% 15N-enriched). Then Cry inclusion bodies were dissolved in urea and purified by Ni column affinity chromatography under denaturing conditions, renatured by dialysis, and further detected by SDS-PAGE and Western blot. The purities of 13C/15N-labeled Cry proteins were each 99%, the amounts of which were 12.6 mg/L and 8.8 mg/L. The δ 13C and δ 15N values of 13C-labeled Cry protein and 15N-labeled Cry protein were 3268.68‰ and 2854.28‰, respectively. An insecticidal test showed that the prokaryotic expression of Cry1Ab/Ac protein had strong insecticidal activity. The stable isotope-labeled insecticidal Cry proteins produced for the first time in this study will provide an experimental basis for future metabolic studies of Cry protein in soil and the characteristics of nitrogen (N) and carbon (C) transformation. The findings will also provide a reference and basis for elucidating the environmental behaviors and ecological effects of Cry plants and expressed products.

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).

Water buffalo (Bubalus bubalus)interferon-αgenes: cloning, expression and antiviral activities

2005 ◽  
Vol 2 (1) ◽  
pp. 45-52
Author(s):  
Shi Xi-Ju ◽  
Xia Chun ◽  
Wang Ming
Keyword(s):  
Recombinant Proteins ◽  
Inclusion Bodies ◽  
Recombinant Expression ◽  
Amino Acid Level ◽  
Viral Disease ◽  
Sds Page ◽  
Pcr Products ◽  
Antiviral Activities ◽  
Expression Plasmids ◽  
Infectious Bovine Rhinotracheitis Virus

AbstractInterferon(IFN)-α genes were cloned from genomic DNA of Fuan and Fuzhong water buffaloes by PCR, and the PCR products were inserted into a pQE30 vector to construct recombinant expression plasmids. Sequence analysis showed that both clones were composed of 498 nucleotides, encoding a mature polypeptide with 166 amino acids (aa). They were defined as two new subtypes, with 91.6–94.2% identity at the amino acid level by comparison with eight previously published bovine IFN-α subtypes. Results of SDS-PAGE and Western blotting showed that each of the recombinant proteins was expressed in inclusion bodies inEscherichia coliwith molecular weight of 20 kDa and the recombinant proteins were 25% of the whole proteins. Inclusion bodies were denatured and renatured with urea and the antiviral activities of the recombinant buffalo IFN-α (rBuIFN-α) were 105U/mg and 106U/mg in CEF/VSV and MDBK/VSV cell lines, respectively. Additionally, rBuIFN-α had good effects against challenge byinfectious bovine rhinotracheitis virus. The rBuIFN-α are potential biological agents for the prevention and treatment of various kinds of bovine viral disease.

Quantitative proteome analysis of cisplatin-induced apoptotic Jurkat T cells by stable isotope labeling with amino acids in cell culture, SDS-PAGE, and LC-MALDI-TOF/TOF MS

2007 ◽  
Vol 28 (23) ◽  
pp. 4359-4368 ◽  
Author(s):  
Frank Schmidt ◽  
Hanne K. Hustoft ◽  
Margarita Strozynski ◽  
Christiane Dimmler ◽  
Thomas Rudel ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Culture ◽  
T Cells ◽  
Stable Isotope ◽  
Isotope Labeling ◽  
Proteome Analysis ◽  
Jurkat T Cells ◽  
Maldi Tof ◽  
Sds Page ◽  
Tof Ms

scholarly journals Recombinant expression and characterization of Solanum tuberosum aspartic protease

SURG Journal ◽  
2009 ◽  
Vol 3 (1) ◽  
pp. 19-25
Author(s):  
Lauren Agro ◽  
Brian Bryska ◽  
Rickey Yada
Keyword(s):  
Solanum Tuberosum ◽  
Disulfide Bonds ◽  
Inclusion Bodies ◽  
Signal Sequence ◽  
Recombinant Expression ◽  
Aspartic Proteinase ◽  
Wild Type ◽  
Sds Page ◽  
Hemoglobin Degradation

Unique to aspartic proteinases from plants are the presence of approximately 100 amino acid regions, which are usually excised during activation of the zymogen. These sequences are termed ‘Plant-Specific Inserts’ and are implicated in membrane interactions of their parent enzymes, including vacuolar targeting and host defense. The need to further characterize the structure-function rol (s) of plant-specific inserts stimulated the current study of the characterization of Solanum tuberosum AP (StAP). Recombinant expression of wild-type StAP resulted in the 54 kDa protein being visualized by SDS-PAGE analysis with a protein yield of 0.03%. A protein purification factor could not be established since activation of the protein at pH 2.2, 3.0, 3.7 and 5.5 was not achieved as evidenced by a lack of change in band patterns on SDS-PAGE as well as acidification and hemoglobin degradation assays. To potentially improve enzyme folding and activation ability, two mutants, (1) lacking the pre-signal sequence and (2) lacking both the signal sequence and the prosegment, were designed, sub-cloned, and expressed. Both products proved to be insoluble and inactive. New constructs were designed for the expression of StAP inclusion bodies for insoluble expression and subsequent re-folding of the protein. Additionally, CysAla mutations for each PSI Cys residue were made to investigate the rol (s) of plant-specific insert disulfide bonds in plant aspartic proteinase enzyme folding and structure. All PSI cysteine mutations (eight mutants) were successfully created using QuickChange MutagenesisTM

Expression of a Fusion Protein of Human Proinsulin with Glutathione-S-Transferase in Escherichia coli

2014 ◽  
Vol 998-999 ◽  
pp. 248-251
Author(s):  
Zhi Xin Di ◽  
Jian Zhong Ma ◽  
Yong Gang Wang
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Inclusion Bodies ◽  
Glutathione S Transferase ◽  
E Coli ◽  
Human Proinsulin ◽  
Sds Page ◽  
Dna Fragment ◽  
Sequence Encoding

A DNA sequence encoding for the human proinsulin was designed according to the codon bias of Escherichia coli and then chemically synthesized. The synthesized DNA fragment was subcloned into pGEX-3X for expression in E. coli BL21 (DE3) and E. coli BL21 Star (DE3), respectively. Conditions for the highest expression of the GST-proinsulin fusion proteins were optimized. These conditions are that cells of E. coli BL21 star (DE3) are incubated in 100mL of the LB medium with 2 mmol/L IPTG and 60μ?g/mL ampicillin at 26oCfor 4h. After disrupted E. coli cells with ultrasonication, inclusion bodies were precipitated from cell lysis and washed. Fusion proteins from the inclusion bodies were redissolved in 8mmol/L of urea. After dialysed in purified water, fusion proteins were analysed by SDS-PAGE. The purity of the fusion protein is about 80.5% in total. The fusion protein from SDS-PAGE was further identified by mass/mass spectrum. GST in the dyad protein is confirmed by the 9 matched sequences. However, the left part is proved a polypeptide of which is completely different from the human proinsulin.

scholarly journals Efficient stable isotope labeling and purification of vitamin D receptor from inclusion bodies

2012 ◽  
Vol 85 (1) ◽  
pp. 25-31 ◽  
Author(s):  
Jinge Zhu ◽  
Hongyu Rao ◽  
Marco Tonelli ◽  
William M. Westler ◽  
Kiran K. Singarapu ◽  
...  
Keyword(s):  
Vitamin D ◽  
Stable Isotope ◽  
Vitamin D Receptor ◽  
Inclusion Bodies ◽  
Isotope Labeling ◽  
Stable Isotope Labeling

scholarly journals Recombinant expression of insoluble enzymes in Escherichia coli: a systematic review of experimental design and its manufacturing implications

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Suraj Mital ◽  
Graham Christie ◽  
Duygu Dikicioglu
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Recombinant Expression ◽  
Holistic Approach ◽  
Protein Product ◽  
Recombinant Enzymes ◽  
E Coli ◽  
Analysis Techniques ◽  
The Impact ◽  
Level Analysis

AbstractRecombinant enzyme expression in Escherichia coli is one of the most popular methods to produce bulk concentrations of protein product. However, this method is often limited by the inadvertent formation of inclusion bodies. Our analysis systematically reviews literature from 2010 to 2021 and details the methods and strategies researchers have utilized for expression of difficult to express (DtE), industrially relevant recombinant enzymes in E. coli expression strains. Our review identifies an absence of a coherent strategy with disparate practices being used to promote solubility. We discuss the potential to approach recombinant expression systematically, with the aid of modern bioinformatics, modelling, and ‘omics’ based systems-level analysis techniques to provide a structured, holistic approach. Our analysis also identifies potential gaps in the methods used to report metadata in publications and the impact on the reproducibility and growth of the research in this field.

Suitable Signal Peptides for Secretory Production of Recombinant Granulocyte Colony Stimulating Factor in Escherichia coli

2020 ◽  
Vol 14 (4) ◽  
pp. 269-282
Author(s):  
Sadra S. Tehrani ◽  
Golnaz Goodarzi ◽  
Mohsen Naghizadeh ◽  
Seyyed H. Khatami ◽  
Ahmad Movahedpour ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Signal Peptide ◽  
Fusion Proteins ◽  
Inclusion Bodies ◽  
Clinical Aspects ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
E Coli ◽  
Secretory Production ◽  
Stimulating Factor

Background: Granulocyte colony-stimulating factor (G-CSF) expressed in engineered Escherichia coli (E. coli) as a recombinant protein is utilized as an adjunct to chemotherapy for improving neutropenia. Recombinant proteins overexpression may lead to the creation of inclusion bodies whose recovery is a tedious and costly process. To overcome the problem of inclusion bodies, secretory production might be used. To achieve a mature secretory protein product, suitable signal peptide (SP) selection is a vital step. Objective: In the present study, we aimed at in silico evaluation of proper SPs for secretory production of recombinant G-CSF in E. coli. Methods: Signal peptide website and UniProt were used to collect the SPs and G-CSF sequences. Then, SignalP were utilized in order to predict the SPs and location of their cleavage site. Physicochemical features and solubility were investigated by ProtParam and Protein-sol tools. Fusion proteins sub-cellular localization was predicted by ProtCompB. Results: LPP, ELBP, TSH, HST3, ELBH, AIDA and PET were excluded according to SignalP. The highest aliphatic index belonged to OMPC, TORT and THIB and PPA. Also, the highest GRAVY belonged to OMPC, ELAP, TORT, BLAT, THIB, and PSPE. Furthermore, G-CSF fused with all SPs were predicted as soluble fusion proteins except three SPs. Finally, we found OMPT, OMPF, PHOE, LAMB, SAT, and OMPP can translocate G-CSF into extracellular space. Conclusion: Six SPs were suitable for translocating G-CSF into the extracellular media. Although growing data indicate that the bioinformatics approaches can improve the precision and accuracy of studies, further experimental investigations and recent patents explaining several inventions associated to the clinical aspects of SPs for secretory production of recombinant GCSF in E. coli are required for final validation.

Conditions Resulting in Formation of Properly Assembled Retroviral Capsids within Inclusion Bodies of Escherichia coli

1999 ◽  
Vol 64 (8) ◽  
pp. 1348-1356 ◽  
Author(s):  
Michaela Rumlová-Kliková ◽  
Iva Pichová ◽  
Eric Hunter ◽  
Tomáš Ruml
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Amorphous Material ◽  
Bacterial Expression ◽  
Biologically Active ◽  
Capsid Assembly ◽  
Cultivation Medium ◽  
Viral Particles ◽  
Induction Temperature

It has been generally accepted that inclusion bodies (IBs) formed in Escherichia coli consist of non-biologically active aggregated proteins, which are stabilized by non-productive interactions. We show here that bacterial expression of a retroviral capsid polyprotein results in formation of insoluble IBs that contain fully assembled viral particles connected with amorphous material. The efficiency of IBs formation and capsid assembly was not significantly affected by changes in induction temperature, pH of cultivation medium or the level of expression.

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