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Validation of Recovery and Purification Processes
Published in James Agalloco, Phil DeSantis, Anthony Grilli, Anthony Pavell, Handbook of Validation in Pharmaceutical Processes, 2021
Protein refolding is an example of a stirred-tank reaction. Overproduced proteins from foreign hosts in bacterial cells are often recovered as refractile or inclusion bodies (IB). The IB are typically 1–3 microns in size and contain (mainly) the protein of interest in a misfolded state. After cell disruption, these dense inclusion bodies are easily separated by centrifugation. Next, the inclusion bodies are washed and solubilized and the proteins refolded to obtain the biologically active product. Solubilization agents are chaotropes such as guani-dine hydrochloride, urea, or sodium thiocyanate; surfactants such as sodium dodecyl sulfate or Triton X-100 are added in the presence of reducing agents. Refolding occurs when the concentration of the solubilization agent is reduced, typically be either dilution or diafiltration, sometimes in the presence of oxidation/reduction reactants. Aggregates may form if the protein concentration is too high. Finally, oxidation of the cysteine residues is needed for allow for correct disulfide bond formation in the native protein.
Selection of Operations in Separation Processes
Published in Juan A. Asenjo, Separation Processes in Biotechnology, 2020
If the intracellular product is manufactured in E. coli, high expression of heterologous proteins will usually accumulate in the form of insoluble inclusion bodies. This makes necessary the processing of the inclusion bodies into the native protein by denaturing and refolding. If the intracellular product is manufactured in yeast, in many instances the protein is present in homogeneous particulate form, typically 30 to 60-nm particles such as viruslike particles (VLPs). Although the processing of intracellular particulate recombinant proteins is an important aspect of downstream process, there are not many satisfactory methods for large-scale separation, dénaturation, and refolding of the particulate proteins. Recent developments in the use of reverse micelles for protein refolding and of two-phase aqueous systems for separation of VLPs from yeast homogenates appear particularly attractive.
Protein Expression Methods
Published in Jay L. Nadeau, Introduction to Experimental Biophysics, 2017
At mid-log phase, protein expression is induced by the addition of IPTG to a final concentration between 0.1 and 2 mM. Lower IPTG concentrations are typically employed for proteins that tend to misfold and are designed to slow protein expression. Rapid protein expression can lead to protein misfolding and the formation of insoluble protein aggregates called inclusion bodies. The Turner bacterial strain provides for the best control of protein expression using IPTG. Induction temperature provides another method to control the rate of protein expression, with the optimal temperature for protein expression typically being between 26°C and 37°C. Finally, induction time provides another method of controlling protein expression. Typical induction times for protein expression range from 2 to 12 h.
Production of bioactive recombinant monoclonal antibody fragment in periplasm of Escherichia coli expression system
Published in Preparative Biochemistry & Biotechnology, 2023
Preeti Saroha, Anurag S. Rathore
Amongst the broadly used expression system, E. coli is the most attractive choice of host for the heterologous proteins. However, due to high expression of recombinant proteins, many form insoluble aggregates within the cytoplasm, thereby resulting in formation of inclusion bodies. The protein in this form is devoid of any functional activity and hence, developing an efficient technique that can express the bioactive protein without compromising the expression level would lessen in the burden on downstream processing and also offer higher process yield. Although there is no universal approach that would be effective for all cases, numerous strategies have been attempted. Single promoter vectors are generally preferred for the expression of recombinant Fabs, with affinity tags for the ease of purification whereas recently upgraded vectors with dual promoters are slowly replacing the former ones due to expression of multi-domain heterologous proteins, such as antibody fragments, rendering a cost effective approach and simultaneously reducing the time of expression.[2,7] Plasmids are categorized based on the copy number as high copy number plasmids (>100) and low copy plasmids (10–12).[2,20]
Bioprocessing of recombinant proteins from Escherichia coli inclusion bodies: insights from structure-function relationship for novel applications
Published in Preparative Biochemistry & Biotechnology, 2023
Kajal Kachhawaha, Santanu Singh, Khyati Joshi, Priyanka Nain, Sumit K. Singh
Although a higher rate of protein synthesis is desirable in therapeutic production, it can often lead to protein aggregate formation in the form of inclusion bodies. When foreign DNA is introduced into E. coli, spatio-temporal regulation of its expression is lost. The recombinant polypeptide is expressed in the E. coli microenvironment, which may not be exactly the same as the source in terms of pH, temperature, redox potential, cofactors, and folding mechanisms. Additionally, in high level expression, hydrophobic sections in the polypeptide are present in large amounts and ready for interaction with related areas during high level expression. All these parameters collectively cause the protein to aggregate.[71]
Improving the soluble expression of aequorin in Escherichia coli using the chaperone-based approach by co-expression with artemin
Published in Preparative Biochemistry and Biotechnology, 2018
Elaheh Khosrowabadi, Zeinab Takalloo, Reza H. Sajedi, Khosro Khajeh
Inclusion body formation of eukaryotic proteins is a common phenomenon when proteins are over-expressed in bacterial hosts.[26] New synthesized polypeptides accumulate as aggregated inclusion bodies and their recovery needs further procedures such as in vitro protein refolding.[27] Co-expression of the susceptible proteins with efficient molecular chaperones in a single host is an effective approach to minimize formation of the inclusion bodies.[28] In the present study, we have used artemin as a molecular chaperone to increase the content of soluble form of aequorin in bacterial cell.