Estimating the potential refolding yield of recombinant proteins expressed as inclusion bodies

2004 ◽  
Vol 87 (5) ◽  
pp. 584-592 ◽  
Author(s):  
Jason G. S. Ho ◽  
Anton P. J. Middelberg
Keyword(s):  
Recombinant Proteins ◽  
Inclusion Bodies

scholarly journals Versatile format of minichaperone-based protein fusion system

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Maria S. Yurkova ◽  
Olga A. Sharapova ◽  
Vladimir A. Zenin ◽  
Alexey N. Fedorov
Keyword(s):  
Recombinant Proteins ◽  
Inclusion Bodies ◽  
Target Protein ◽  
Fusion System ◽  
Protein Fusion ◽  
Gene Engineering ◽  
Apical Domain ◽  
Stable Forms ◽  
Protein Variants ◽  
Protein Moiety

Abstract Hydrophobic recombinant proteins often tend to aggregate upon expression into inclusion bodies and are difficult to refold. Producing them in soluble forms constitutes a common bottleneck problem. A fusion system for production of insoluble hydrophobic proteins in soluble stable forms with thermophilic minichaperone, GroEL apical domain (GrAD) as a carrier, has recently been developed. To provide the utmost flexibility of the system for interactions between the carrier and various target protein moieties a strategy of making permutated protein variants by gene engineering has been applied: the original N- and C-termini of the minichaperone were linked together by a polypeptide linker and new N- and C-termini were made at desired parts of the protein surface. Two permutated GrAD forms were created and analyzed. Constructs of GrAD and both of its permutated forms fused with the initially insoluble N-terminal fragment of hepatitis C virus’ E2 protein were tested. Expressed fusions formed inclusion bodies. After denaturation, all fusions were completely renatured in stable soluble forms. A variety of permutated GrAD variants can be created. The versatile format of the system provides opportunities for choosing an optimal pair between particular target protein moiety and the best-suited original or specific permutated carrier.

scholarly journals Recovery of recombinant proteins CFP10 and ESAT6 from Escherichia coli inclusion bodies for tuberculosis diagnosis: a statistical optimization approach

2019 ◽  
Vol 3 (2) ◽  
pp. 298-305 ◽  
Author(s):  
Ludmilla Dela Coletta Troiano Araujo ◽  
Daniel Ernesto Rodriguez-Fernández ◽  
Márcia Wibrantz ◽  
Susan Grace Karp ◽  
Gilberto Delinski Junior ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Inclusion Bodies ◽  
Statistical Optimization ◽  
Optimization Approach ◽  
Tuberculosis Diagnosis

Capillary gel electrophoresis for the quantification and purity determination of recombinant proteins in inclusion bodies

2013 ◽  
Vol 34 (18) ◽  
pp. 2754-2759 ◽  
Author(s):  
Carlos E. Espinosa-de la Garza ◽  
Francisco C. Perdomo-Abúndez ◽  
Víctor R. Campos-García ◽  
Néstor O. Pérez ◽  
Luis F. Flores-Ortiz ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Gel Electrophoresis ◽  
Inclusion Bodies ◽  
Purity Determination ◽  
Capillary Gel Electrophoresis

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.

Expression, solubilization, refolding and final purification of recombinant proteins as expressed in the form of "classical inclusion bodies" in E. coli

2020 ◽  
Vol 27 ◽  
Author(s):  
Mohammad Sadegh Hashemzadeh ◽  
Mozafar Mohammadi ◽  
Hadi Esmaeili Gouvarchin Ghaleh ◽  
Mojtaba Sharti ◽  
Ali Choopani ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Inclusion Bodies ◽  
Native Conformation ◽  
Native Form ◽  
Over Expression ◽  
E Coli ◽  
Mild Conditions ◽  
Final Purification

: Escherichia coli has been most widely used for production of the recombinant proteins. Over-expression of the recombinant proteins is the mainspring of the inclusion bodies formation. The refolding of these proteins into bioactive forms is cumbersome and partly time-consuming. In the present study, we reviewed and discussed most issues regarding the recovery of "classical inclusion bodies" by focusing on our previous experiences. Performing proper methods of expression, solubilization, refolding and final purification of these proteins, would make it possible to recover higher amounts of pro-teins into the native form with appropriate conformation. Generally, providing mild conditions and proper refolding buffers, would lead to recover more than 40% of inclusion bodies into bioactive and native conformation.

Comparative study to develop a single method for retrieving wide class of recombinant proteins from classical inclusion bodies

2018 ◽  
Vol 102 (5) ◽  
pp. 2363-2377 ◽  
Author(s):  
Arshad Ahmed Padhiar ◽  
Warren Chanda ◽  
Thomson Patrick Joseph ◽  
Xuefang Guo ◽  
Min Liu ◽  
...  
Keyword(s):  
Comparative Study ◽  
Recombinant Proteins ◽  
Wide Class ◽  
Inclusion Bodies ◽  
Single Method

scholarly journals Nucleic Acids In Inclusion Bodies Obtained From E. Coli Cells Expressing Human Interferon-Gamma

2020 ◽  
Author(s):  
Elena Krachmarova ◽  
Ivan Ivanov ◽  
Genoveva Nacheva
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Nucleic Acids ◽  
Interferon Gamma ◽  
Recombinant Proteins ◽  
Gel Electrophoresis ◽  
Inclusion Bodies ◽  
Protein Aggregates ◽  
Human Interferon ◽  
Molecular Heterogeneity

Abstract BackgroundInclusion bodies (IBs) are protein aggregates in recombinant bacterial cells containing mainly the target recombinant protein. Although it has been shown that IBs contain functional proteins along with protein aggregates, their direct application as pharmaceuticals is hindered by their heterogeneity and hazardous contaminants with bacterial origin. Therefore, together with the production of soluble species, IBs remain the main source for manufacture of recombinant proteins with medical application. The quality and composition of the IBs affect the refolding yield and further purification of the recombinant protein. The knowledge whether nucleic acids are genuine components or concomitant impurities of the IBs is a prerequisite for the understanding of the IBs formation and for development of optimized protocols for recombinant protein refolding and purification. IBs isolated from Escherichia coli overexpressing human interferon-gamma (hIFNγ), a protein with therapeutic application, were used as a model. ResultsIBs were isolated from Escherichia coli LE392 cells transformed with a hIFNγ expressing plasmid under standard conditions and further purified by centrifugation on a sucrose cushion, followed by several steps of sonication and washings with non-denaturing concentrations of urea. The efficiency of the purification was estimated by SDS-PAGE gel electrophoresis and parallel microbiological testing for the presence of residual intact bacteria. Phenol/chloroform extraction showed that the highly purified IBs contain both DNA and RNA. The latter were studied by UV spectroscopy and agarose gel electrophoresis combined with enzymatic treatment and hybridization. DNA was observed as a diffuse fraction mainly in the range of 250 to 1000 bp. RNA isolated by TRIzol® also demonstrated a substantial molecular heterogeneity. Hybridization with 32P-labelled oligonucleotides showed that the IBs contain rRNA and are enriched of hIFNγ mRNA.ConclusionsThe results presented in this study indicate that the nucleic acids might be intrinsic components rather than co-precipitated impurities in the IBs. We assume that the nucleic acids are active participants in the aggregation of recombinant proteins and formation of the IBs that originate from the transcription and translation machinery of the microbial cell factory. Further studies are needed to ascertain this notion.

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