Solid-phase refolding of poly-lysine tagged fusion protein of hEGF and angiogenin

A fusion protein, consisting of a human epidermal growth factor (hEGF) as the recognition domain and human angiogenin as the toxin domain, can be used as a targeted therapeutic against breast cancer cells among others. The fusion protein was expressed as inclusion body in recombinantE. coli, and when the conventional, solution-phase refolding process was used the refolding yield was very low due to severe aggregation. It was probably because of the opposite electric charge at a neutral pH resulting from the vastly different pI values of each domain. The solidphase refolding process that exploited the ionic interactions between ionic exchanger surface and the fusion protein was tried, but the adsorption yield was also very low, below 30%, regardless of the resins and pH conditions used. Therefore, to provide a higher ionic affinity toward the solid matrix, six lysine residues were tagged to theN-terminus of the hEGF domain. When heparin-Sepharose was used as the matrix, the adsorption capacity increased 2.5–3 times to about 88%. Besides the intrinsic affinity of angiogenin to heparin the poly-lysine tag provided additional ionic affinity. And the subsequent refolding yield increased nearly 13-fold, fromc.a 4.8% in the conventional refolding of the untagged fusion protein to 63.6%. The process was highly reproducible. The refolded protein in the column eluate retained R Nase bioactivity, of angiogenin.     

A Comparative Investigation on Different Refolding Strategies of Recombinant Human Tissue-type Plasminogen Activator Derivative

Recombinant human tissue-type plasminogen activator derivative (r-PA), fused with thioredoxin (Trx), was expressed in Escherichia coli. The resultant fusion protein, Trx-r-PA, was almost completely in the form of inclusion bodies and without activity. Different refolding strategies were investigated including different post-treatment of solubilized Trx-r-PA inclusion bodies, on-column refolding by size-exclusion chromatography (SEC) using three gel types (Sephacryl S-200, S-300 and S-400), refolding by Sephacryl S-200 with a urea gradient and two-stage temperature control in refolding. An optimized on-column refolding process for Trx-r-PA inclusion bodies was established. The collected Trx-r-PA inclusion bodies were dissolved in 6 m guanidine hydrochloride (Gdm·HCl), and the denatured protein was separated from dithiothreitol (DTT) and Gdm·HCl with a G25 column and simultaneously dissolved in 8 m urea containing oxidized glutathione (GSSG). Finally a refolding of Trx-r-PA protein on Sephacryl S-200 column with a decreasing urea gradient combined with two-stage temperature control was employed, and the activity recovery of refolded protein was increased from 3.6 to 13.8% in comparison with the usual dilution refolding. Revisions requested 31 October 2005; Revisions received 20 December 2005     

SOn-column Protein Refolding for Crystallization

One major bottleneck in protein production in Escherichia coli for structural genomics projects is the formation of insoluble protein aggregates (inclusion bodies). The efficient refolding of proteins from inclusion bodies is becoming an important tool that can provide soluble native proteins for structural and functional studies. Here we report an on-column refolding method established at the Berkeley Structural Genomics Center (BSGC). Our method is a combination of an ‘artificial chaperone-assisted refolding’ method previously proposed and affinity chromatography to take advantage of a chromatographic step: less time-consuming, no filtration or concentration, with the additional benefit of protein purification. It can be easily automated and formatted for high-throughput process.     

One-step Purification and Immobilization in Cellulose of the GroEL Apical Domain Fused to a Carbohydrate-binding Module and its use in Protein Refolding

The apical domain of the chaperonin, GroEL, fused to the carbohydrate binding module type II, CBD_Cex, of Cellulomonas fimi, was expressed in Escherichia coli. The recombinant protein, soluble or from inclusion bodies, was directly purified and immobilized in microcrystalline cellulose particles or cellulose fabric membranes. Assisted refolding of rhodanese by the immobilized mini-chaperone showed a two-fold improvement as compared to a control. Using chromatographic refolding, 35% of rhodanese activity was recovered in only 5 min (mean residence time) as compared to 17% for spontaneous refolding. This mini-chaperone immobilized in cellulose could be a cost-efficient method to refold recombinant proteins expressed as inclusion bodies.     

Refolding of the Fusion Protein of Recombinant Enterokinase Light Chain rEKL

The fusion protein of enterokinase light chain, DsbA-rEK_L, was expressed mainly in the inclusion body in E. coli. The recombinant bacteria were fermented to high density, with high expression of the fusion protein. After being washed with 0.5 % Triton X-100 and 4 mol/L urea, the inclusion body was dissolved in 6 mol/L guanidine and 100 mmol/L DTT, derivatized by cystine and refolded by pulse refolding. The strategy of pulse refolding involved the addition of 0.03 mg/mL of fusion protein until its final concentration reached 0.3 mg/mL. The refolded protein was autocleaved, and the active EK_L molecule was released after the addition of 2 mmol/L of CaCl₂. Using the two-step purification processes of IDA-Sepharose chromatography and Q-Sepharose chromatography, the purity of rEK_L was found to be above 95 %, with a high activity to cleave the recombinant reteplase fusion protein, Trx-rPA. The yield of purified rEK_L was more than 60 mg/L of cultures. As a result, the therapeutic proteins like rPA could be produced on a large scale in a way such as expressed in the form of fusion proteins.     

A suitable and effective stepwise oxidative refolding procedure for highly-cationic tetrameric avidin in nucleic acid free conditions

Optimized conditions are needed to refold recombinant proteins from bacterial inclusion bodies into their biologically active conformations. In this study, we found two crucial requirements for efficient refolding of cationic tetrameric chicken avidin. The first step is to eliminate nucleic acid contaminants from the bacterial inclusion body. The electrostatic interactions between the remaining nucleic acids and proteins strongly enhanced protein aggregation during the refolding process. The cysteine specific reversible S-cationization procedure was successfully employed for large-scale preparation of nucleic acid free denatured protein without purification tag system. The second step is the intramolecular disulfide formation prior to refolding in dialysis removing denaturant. Disulfide intact monomeric avidin showed efficient formation of biologically active tetrameric conformation during the refolding process. Using this optimized refolding procedure, highly cationic avidin derivative designed as an intracellular delivery carrier of biotinylated protein was successfully prepared.     

Refolding of superoxide dismutase by ion-exchange chromatography

A new ion-exchange chromatography process was developed for refolding of iron superoxide dismutase (Fe-SOD) produced in Escherichia coli as an inclusion body. After adsorption on an ion-exchange matrix, the denatured protein was eluted by gradient decrease of urea concentration and pH of the elution buffer. The dual gradient allowed the denatured protein to refold to its correct native conformation with return of biological activity. Compared with the traditional dilution, refolding process, the new process increased the refolding yield five-fold. The process could also be carried out at high protein concentration to decrease the solution volume after refolding.     

huGM-CSF(9-127)-IL-6(29-184)融合蛋白的复性及纯化研究

利用Q Sepharose H.P.离子交换柱层析在8mol/L尿素变性条件下对huGM-CSF(9-127)-IL-6(29-184）融合蛋白进行初步纯化,然后再利用Sephacryl S-200分子筛柱层析复性及纯化后获得目的蛋白,其纯度达到95％以上。该纯化方案成功地解决了稀释复性或透析复性产物在进行Q Sepharose H.P.离子交换柱层析时目的蛋白不稳定而沉积于柱上的问题,获得了较好的复性效果,复性率达到80％以上。使用该纯化方案,1天内便可基本完成重组蛋白的复性及纯化过程,而且也便于扩大。     

Improved Recovery of a Fusion Protein Containing the Antigenic Domain 1 of the Human Cytomegalovirus Glycoprotein B

Heterologous expression in Escherichia coli often leads to production of the expressed proteins as insoluble and inactive inclusion bodies. The general strategy for protein recovery includes isolation and washing of inclusion bodies, solubilization of aggregated protein and refolding of solubilized protein. The process of refolding, as well as the other steps involved in inclusion body recovery, must be optimized according to the characteristics of each protein. For the development of reliable and inexpensive serodiagnostic tests, the antigenic domain 1 (AD-1) of human cytomegalovirus glycoprotein B was expressed in E. coli and a process was developed to increase recovery of the fusion protein containing AD-1. A comparison of disruption methods and different conditions involved in recovery of this fusion protein from inclusion bodies is presented. The developed method gives a high yield of the fusion protein with a purity sufficient for use in diagnostic tests.     

Protein folding in vivo and renaturation of recombinant proteins from inclusion bodies

Eukaryotic proteins expressed inEscherichia coli often accumulate within the cell as insoluble protein aggregates or inclusion bodies. The recovery of structure and activity from inclusion bodies is a complex process, there are no general rules for efficient renaturation. Research into understanding how proteins fold in vivo is giving rise to potentially new refolding methods, for example, using molecular chaperones. In this article we review what is understood about the main three classes of chaperone: the Stress 60, Stress 70, and Stress 90 proteins. We also give an overview of current process strategies for renaturing inclusion bodies, and report the use of novel developments that have enhanced refolding yields.     

A Combined Refolding Technique for Recombinant Human Interferon-γ Inclusion Bodies by Ion-exchange Chromatography with a Urea Gradient

Summary A refolding strategy was described for on-column refolding of recombinant human interferon-γ (rhIFN-γ) inclusion bodies by ion-exchange chromatography (IEC). The rhIFN-γ was expressed in E. colias inclusion bodies. Triton X-100 was used first to wash the rhIFN-γ inclusion bodies before chromatographic refolding. The refolding process was performed by gradually decreasing the concentration of urea in the column after the denatured rhIFN-γ protein had bound onto the ion-exchange gel SP-Sepharose Fast Flow. The refolding and purification process for the denatured rhIFN-γ was carried through simultaneously and the purity of the refolded rhIFN-γ was up to 95%. The effects of protein loading, flow rate, urea gradient length and final urea concentration on the refolding were investigated in detail. Under the optimum conditions, the specific activity of rhIFN-γ was up to 7.5 × 10⁵ IU mg⁻¹and active protein recovery was up to 54%.     

Refolding of fusion ferritin by gel filtration chromatography (GFC)

Fusion ferritin (heavy chain ferritin, F_H+light chain ferritin, F_L), an iron-binding protein, was primarily purified from recombinantEscherichia coli by two-step sonications with urea [1]. Unfolded ferritin was refolded by gel filtration chromatography (GFC) with refolding enhancer, where 50 mM Na-phosphate (pH 7.4) buffer containing additives such as Tween 20, PEG, andl-arginine was used. Ferritin is a multimeric protein that contains approximately 20 monomeric units for full activity. Fusion ferritin was expressed in the form of inclussion bodies (Ibs). The IBs were initially solubilized in 4 M urea denaturant. The refolding process was then performed by decreasing the urea concentration on the GFC column to form protein multimers. The combination of the buffer-exchange effect of GFC and the refolding enhancers in refolding buffer resulted in an efficient route for producing properly folded fusion ferritin.     

Optimized procedures for producing biologically active chemokines

We describe here two strategies to produce biologically active chemokines with authentic N-terminal amino acid residues. The first involves producing the target chemokine with an N-terminal 6×His-SUMO tag in Escherichia coli as inclusion bodies. The fusion protein is solubilized and purified with Ni–NTA–agarose in denaturing reagents. This is further followed by tag removal and refolding in a redox refolding buffer. The second approach involves expressing the target chemokine with an N-terminal 6×His-Trx-SUMO tag in an engineered E. coli strain that facilitates formation of disulfide bonds in the cytoplasm. Following purification of the fusion protein via Ni–NTA and tag removal, the target chemokine is refolded without redox buffer and purified by reverse phase chromatography. Using the procedures, we have produced more than 15 biologically active chemokines, with a yield of up to 15 mg/L.     

Predictions of matrix‐assisted refolding of α‐lactalbumin: Process efficiency versus batch dilution method

Protein refolding is an important technique to produce active recombinant proteins from inclusion bodies. Because of the complexity of the refolding process, a trial‐and‐error method is usually used for its design, which is ineffective and time consuming. Therefore, an efficient method for the process prediction is indispensable to optimize the operating conditions. In this article, we suggest a design procedure for matrix‐assisted protein refolding. Three different chromatographic techniques were considered exploiting hydrophobic interaction chromatography, ion‐exchange chromatography, and SEC media. The procedure consisted of quantification of refolding kinetics, analysis of the retention behavior of all protein forms involved in refolding, construction of a dynamic model, and the process simulation. Denatured bovine α‐lactalbumin was used as model protein. The refolding rate was measured for different protein concentration using the batch dilution method. A kinetic scheme for the protein refolding was suggested and incorporated into a dynamic model of chromatographic column and used for predicting the refolding performance. The productivity, yield, and buffer consumption were used as performance indicators for the refolding techniques considered. The matrix‐assisted protein refolding process outperformed batch dilution method with respect to all indicators provided that efficient method for the process design was used.     

Isotopically labeled expression in E. coli, purification, and refolding of the full ectodomain of the influenza virus membrane fusion protein

This paper describes methods to produce an isotopically labeled 23 kDa viral membrane protein with purified yield of 20 mg/L of Escherichia coli shake flask culture. This yield is sufficient for NMR structural studies and the protein production methods are simple, straightforward, and rapid and likely applicable to other recombinant membrane proteins expressed in E. coli. The target FHA2 protein is the full ectodomain construct of the influenza virus hemagglutinin protein which catalyzes fusion between the viral and the cellular endosomal membranes during infection. The high yield of FHA2 was achieved by: (1) initial growth in rich medium to A₆₀₀  8 followed by a switch to minimal medium and induction of protein expression; and (2) obtaining protein both from purification of the detergent-soluble lysate and from solubilization, purification, and refolding of inclusion bodies. The high cell density was achieved after optimization of pH, oxygenation, and carbon source and concentration, and the refolding protocol was optimized using circular dichroism spectroscopy. For a single residue of membrane-associated FHA2 that was obtained from purification and refolding of inclusion bodies, native conformation was verified by the ¹³CO chemical shifts measured using solid-state nuclear magnetic resonance spectroscopy.     

Refolding and purification of recombinant human interferon-γ expressed as inclusion bodies inEscherichia coli using size exclusion chromatography

A size exclusion chromatography (SEC) process, in the presence of denaturant in the refolding buffer was developed to refold recombinant human interferon-γ (rhIFN-γ) at a high concentration. The rhIFN-γ was overexpressed inE. coli, resulting in the formation of inactive inclusion bodies (IBs). The IBs were first solubilized in 8 M urea as the denaturant, and then the refolding process performed by decreasing the urea concentration on the SEC column to suppress protein aggregation. The effects of the urea concentration, protein loading mode and column height during the refolding step were investigated. The combination of the bufferexchange effect of SEC and a moderate urea concentration in the refolding buffer resulted in an efficient route for producing correctly folded rhIFN-γ, with protein recovery of 67.1% and specific activity up to 1.2×10⁷ IU/mg.     

融合牛肠激酶轻链EKL包涵体蛋白的复性纯化

大肠杆菌高密度发酵表达肠激酶轻链融合蛋白DsbA-rEKL,主要以包涵体形式存在。包涵体经4mol/L尿素和0.5%TritonX-100洗涤,以6mol/L盐酸胍、100mmol/LDTT溶解,在胱氨酸存在下,以脉冲加样方式复性。融合蛋白复性在6mmol/L胱氨酸存在下、脉冲加量0.03mg/mL和复性终蛋白浓度0.3mg/mL为最佳复性方案。复性的融合蛋白加2mmol/LCaCL2后快速自切。经IDA-Sepharose及Q-Sepharose纯化,rEKL纯度可达95%以上,可高效酶切重组瑞特普酶融合蛋白Trx-rPA。实现了大规模生产rEKL,每升发酵液经复性及纯化后,可得rEKL60mg/L以上,使以融合蛋白表达rPA等药用蛋白成为现实。     

Refolding of Npro fusion proteins

The autoprotease N^pro significantly enhances expression of fused peptides and proteins and drives the formation of inclusion bodies during protein expression. Upon refolding, the autoprotease becomes active and cleaves itself specifically at its own C‐terminus releasing the target protein with its authentic N‐terminus. N^pro wild‐type and its mutant EDDIE, respectively, were fused N‐terminally to the model proteins green fluorescent protein, staphylococcus Protein A domain D, inhibitory peptide of senescence‐evasion‐factor, and the short 16 amino acid peptide pep6His. In comparison with the N^pro wild‐type, the tailored mutant EDDIE displayed an increased rate constant for refolding and cleavage from 1.3 × 10^?4 s^?1 to 3.5 × 10^?4 s^?1, and allowed a 15‐fold higher protein concentration of 1.1 mg/mL when studying pep6His as a fusion partner. For green fluorescent protein, the rate constant was increased from 2.4 × 10^?5 s^?1 to 1.1 × 10^?4 s^?1 when fused to EDDIE. When fused to small target peptides, refolding and cleavage yields were independent of initial protein concentration, even at high concentrations of 3.9 mg/mL, although cleavage rates were strongly influenced by the fusion partner. This behavior differed from conventional 1st order refolding kinetics, where yield strongly depends on initial protein concentration due to an aggregation reaction of higher order. Refolding and cleavage of EDDIE fusion proteins follow a monomolecular reaction for the autoproteolytic cleavage over a wide concentration range. At high protein concentrations, deviations from the model assumptions were observed and thus smaller rate constants were required to approximate the data. Biotechnol. Bioeng. 2009; 104: 774–784 © 2009 Wiley Periodicals, Inc.     

Heterologous expression of the <Emphasis Type="Italic">msp2 gene</Emphasis> from <Emphasis Type="Italic">Marasmius scorodonius</Emphasis>

For the heterologous expression of the msp2 gene from the edible mushroom Marasmius scorodonius in Escherichia coli the cDNA encoding the extracellular Msp2 peroxidase was cloned into the pBAD III expression plasmid. Expression of the protein with or without signal peptide was investigated in E. coli strains TOP10 and LMG194. Different PCR products were amplified for expression of the native target protein or a protein with a signal peptide. Omitting the native stop codon and adding six His-residues resulted in a fusion protein amenable to immune detection and purification by immobilised metal affinity chromatography. In E. coli the recombinant protein was produced in high yield as insoluble inclusion bodies. The influence of different parameters on MsP2 refolding was investigated. Active enzyme was obtained by glutathione-mediated oxidation in a medium containing urea, Ca²⁺, and hemin.     

Integrated continuous processing of proteins expressed as inclusion bodies: GCSF as a case study

Affordability of biopharmaceuticals continues to be a challenge, particularly in developing economies. This has fuelled advancements in manufacturing that can offer higher productivity and better economics without sacrificing product quality in the form of an integrated continuous manufacturing platform. While platform processes for monoclonal antibodies have existed for more than a decade, development of an integrated continuous manufacturing process for bacterial proteins has received relatively scant attention. In this study, we propose an end‐to‐end integrated continuous downstream process (from inclusion bodies to unformulated drug substance) for a therapeutic protein expressed in Escherichia coli as inclusion body. The final process consisted of a continuous refolding in a coiled flow inverter reactor directly coupled to a three‐column periodic counter‐current chromatography for capture of the product followed by a three‐column con‐current chromatography for polishing. The continuous bioprocessing train was run uninterrupted for 26 h to demonstrate its capability and the resulting output was analyzed for the various critical quality attributes, namely product purity (>99%), high molecular weight impurities (<0.5%), host cell proteins (<100 ppm), and host cell DNA (<10 ppb). All attributes were found to be consistent over the period of operation. The developed assembly offers smaller facility footprint, higher productivity, fewer hold steps, and significantly higher equipment and resin utilization. The complexities of process integration in the context of continuous processing have been highlighted. We hope that the study presented here will promote development of highly efficient, universal, end‐to‐end, fully continuous platforms for manufacturing of biotherapeutics. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:998–1009, 2017