Inclusion Body Washing Research Articles

Purification technology design, biochemical and immunological characteristics of the recombinant chimeric antigen for evaluation of T cell immunity against coronavirus infection

Diagnosis of specific T cell immunity to the antigenic determinants of SARS-CoV-2 in patients seems to be an increasingly important task due to accumulation of data about the role of T cell immune response in course of coronavirus infection and clearance of SARS-CoV-2 in case of secondary infection. Previously, we designed the recombinant CorD_PS antigen for evaluation of T cell antiviral immunity, containing conservative and immunogenic sequences of structural proteins of the SARS-CoV-2 coronavirus. E. coli CorD_PS producing strain with stable expression of the recombinant CorD_PS antigen was obtained. Aim of the present work is to design a laboratory technology for the production of recombinant antigen CorD_PS, to control the quality of the obtained chimeric protein and to study its immunological activity. Development of the cultivation conditions for E. coli CorD_PS cells was carried out in conical flasks in LB-M_Km medium at 37 °C, then scaled in a fermenter with a volume of 30 liters. Expression was induced by the addition of IPTG. Expression was controlled in culture lysates in 12% SDS-PAGE. The resulting biomass was lysed using an ultrasonic disintegrator with followed by centrifugation. The compositions of lysing and solubilizing buffers were selected, as well as conditions for refolding of the recombinant protein. Cation exchange (SP-sepharose), hydrophobic (Butylsepharose) and exclusive (Sephacryl S-200 HR) chromatography were used sequentially to purify the dissolved protein. Protein impurities in the preparation were determined by reverse-phase HPLC and electrophoresis in 12% SDS-PAGE, residual lipopolysaccharides were determined using a gel-thrombin variant of the LAL test, residual proteins of the producer strain were determined by enzyme immunoassay, residual DNA of the producer strain was determined by binding with PicoGreen dye. Specificity was controlled by indirect enzyme immunoassay. The evaluation of cytokine production by CD4+T lymphocytes in response to their stimulation by recombinant antigen ex vivo was performed on a flow cytofluorimeter. The biomass yield during cultivation of E. coli CorD_PS in a 30L fermenter was up to 20 g/L for 4 hours of 0.1 mM IPTG induction. Sequential washing of inclusion bodies from bacterial cellular components and their subsequent solubilization in a buffer containing 8 M urea allowed to obtain a solution of denatured antigen with a concentration of 10 mg/mL. The efficiency of refolding by dilution was 75%. After three stages of chromatographic purification, protein samples with a concentration of 1.2-1.4 mg/mL, HPLC purity of 98.43%, corresponding to key quality parameters according to the OFS.1.7.1.0007.15, were obtained. The recombinant antigen showed specific binding to a sample of the First WHO International Standard and sample Company Reference Standard No. 3 of anti- SARS-CoV-2 human immunoglobulins in a specified concentration range. CD4+T lymphocytes effectively responded to recombinant antigen treatment by increasing IFNγ production. The optimal concentration of recombinant coronavirus antigen was 5 μg/mL. The developed technological process makes it possible to obtain 5-7 grams of coronavirus recombinant CorD_PS antigen in one cultivation cycle in 30 liters of fermentation medium with key quality parameters according to the OFS.1.7.1.0007.15. As a result of specific immunological activity studies of the recombinant coronavirus antigen CorD_PS, the concept of its possible use as a diagnostic tool for determining the formation of a T cell immune response was confirmed.

New and efficient purification process for recombinant human insulin produced in Escherichia coli.

A new and efficient purification process for recombinant human insulin production was developed by exploring new resins and optimizing purification steps from E. coli inclusion body washing to insulin polishing. A combined additives inclusion body wash protocol drastically improved efficiency in clarifying ZZ-proinsulin samples. ZZ-proinsulin recovery increased three-fold under optimized solubilization and sulfitolysis incubation temperature and duration. Desalting with Bio-Gel P4 and P6 resulted in higher sample loading and product recovery compared to conventional resins. A higher recovery (96%) and purity (81%) of ZZ-proinsulin were achievable with the Nuvia S cation exchanger for proinsulin purification compared to a reported process using expensive affinity chromatography resin. As the first step for insulin purification, process scale-up is more economical and practical when Nuvia HR-S cation exchanger was used instead of commonly used reversed-phase chromatography. Nuvia HR-S was highly effective in removing ZZ fusion protein (90% removal) after enzymatic cleavage, although ZZ fusion protein has a very close theoretical pI to human insulin, which was supposedly challenging to be removed by cation exchange chromatography. Also, insulin can be eluted at a lower ethanol % using Nuvia HR-S compared to other reported processes and is thus more environmentally sustainable. Recombinant human insulin was obtained with over 98% purity in just a single reversed-phase polishing step, which is comparable to the reference standard. The process workflow presented here can be potentially applied for the development of purification workflow for insulin analogs or other peptide products derived from E. coli inclusion body.Key points• Drastic efficiency improvement for inclusion body wash with combined additives.• High recovery of proinsulin purification with high capacity cation exchange resin.• Effective removal of fusion tag at lower ethanol % with high-resolution resin.

Development of recombinant human granulocyte colony-stimulating factor (nartograstim) production process in Escherichia coli compatible with industrial scale and with no antibiotics in the culture medium.

The granulocyte colony-stimulating factor (G-CSF) is a hematopoietic cytokine that has important clinical applications for treating neutropenia. Nartograstim is a recombinant variant of human G-CSF. Nartograstim has been produced in Escherichia coli as inclusion bodies (IB) and presents higher stability and biological activity than the wild type of human G-CSF because of its mutations. We developed a production process of nartograstim in a 10-L bioreactor using auto-induction or chemically defined medium. After cell lysis, centrifugation, IB washing, and IB solubilization, the following three refolding methods were evaluated: diafiltration, dialysis, and direct dilution in two refolding buffers. Western blot and SDS-PAGE confirmed the identity of 18.8-kDa bands as nartograstim in both cultures. The auto-induction medium produced 1.17 g/L and chemically defined medium produced 0.95 g/L. The dilution method yielded the highest percentage of refolding (99%). After refolding, many contaminant proteins precipitated during pH adjustment to 5.2, increasing purity from 50 to 78%. After applying the supernatant to cation exchange chromatography (CEC), nartograstim recovery was low and the purity was 87%. However, when the refolding solution was applied to anion exchange chromatography followed by CEC, 91%-98% purity and 2.2% recovery were obtained. The purification process described in this work can be used to obtain nartograstim with high purity, structural integrity, and the expected biological activity. KEY POINTS: • Few papers report the final recovery of the purification process from inclusion bodies. • The process developed led to high purity and reasonable recovery compared to literature. • Nartograstim biological activity was demonstrated in mice using a neutropenia model.

Способ получения рекомбинантного гранулоцитарно-макрофагального колониестимулирующего фактора человека

A suggested method for obtaining recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) includes the accumulation of the producer strain biomass enriched with the target product up to 30% of total protein content, its isolation and purification. The later consists of the following stages: ultrasound cell disintegration, washing of inclusion bodies with buffer solutions, GM-CSF solubilization from inclusion bodies by 6 M urea, denaturation-renaturation of protein molecules and purification by chromatography on DEAE-Sepharose and combined chromatography on CM-Sepharose and Q-Sepharose followed by dialysis. The proposed method makes it possible to yield up to 10 mg of the protein preparation from 1 g of wet cells with the purity of 98% and high activity shown on the human erythroleukemia cell line. granulocyte-macrophage colony-stimulating, GM-CSF, producer strain, cultivation, chromatographic purification. The work was performed in the framework of the State Assignment «Adjustment of the Technology of Preparative Obtaining and Purification of Recombinant Proteins» (no. 13/18).

Immunogenicity and immunoreactivity of Tp0821 recombinant protein from Treponema pallidum

Treponemapallidum (Tp) is responsible for invading reproductive organs and the skin in early stages, and involves almost all organs/systems at advanced stages. In the present study, screening of the dominant epitope fragment of the Tp outer membrane protein, Tp0821, was performed and the prokaryotic expression vector pQE32/Tp0821 was constructed. The denaturation and dialysis of rTp0821 were achieved through ultrasound, inclusion body washing and dissolution. Experiments in purified rTp0821‑immune New Zealand rabbits indicated that the recombinant proteins were of high immunogenicity, and the irritation led a marked humoral immune response in the New Zealand rabbits. Western blot analysis showed that the purified recombinant proteins reacted with the Tp‑positive infected serum, confirming the high level of immunoreactivity. The delayed type hypersensitivity of rTp0821 recombinant proteins was positive, indicating that rTp0821 induced a specific cell immune response and was selected as a Tp vaccine candidate protein. The findings of the present study provided novel evidence, which provided information for further investigations on the pathogenic mechanism of Tp and the development of diagnostic reagents.

Preparative method for obtaining recombinant human interferon α2b from inclusion bodies of Escherichia coli

A simple, easily reproducible, and scalable method for obtaining recombinant human interferon α2b from Escherichia coli inclusion bodies has been elaborated. It involves the following steps: preparation of producer cell biomass, isolation and washing of inclusion bodies, their dissolution with protein refolding, SP Sepharose chromatography, and DEAE Sepharose chromatography. According to the results of gel electrophoresis and reversed-phase HPLC, the purity of the protein obtained exceeds 95%.

CaCl<sub>2</sub>/EDTA 및 비이온성 계면활성제 활용 Inclusion Body 정제법을 이용한 BA-RGD 단백질의 생산

The limited productivity of natural shell matrix proteins has hampered the investigation of their biochemical properties and practical applications, although biominerals in nature obtained by organic-inorganic assemblies have attractive mechanical and biological properties. Here, we prepared a vector for the expression of a fusion protein of a shell matrix protein from Pinctada fucata (named as GRP_BA) with the GRGDSP residue. The fusion protein of BA-RGD was simply produced in E. coli and purified through sequential steps including the treatment with CaCl₂ and EDTA solution for cell membrane washing, mechanical cell disruption and the application of non-ionic surfactant of Triton X-100 for BA-RGD inclusion body washing. The production yield was approximately 60 mg/L, any other protein band was not observed in SDS-PAGE and it was estimated that above 97% endotoxin was removed compared to the endotoxin level of whole cell. This study showed this simple and easy purification approach could be applied to the purification of BA-RGD fusion protein. It is expected that the protein could be utilized for the preparation of biominerals in practical aspects.

Improving the mda-7/IL-24 refolding and purification process using optimized culture conditions based on the structure characteristics of inclusion bodies

BackgroundThe melanoma differentiation-associated gene-7 (mda-7)/interleukin-24 (IL-24) can induce apoptosis in a wide variety of tumor cell types, whereas it has no toxicity in normal cells. However, recombinant human mda-7/IL-24 is difficult to obtain from Escherichia coli because of its insolubility.ResultsIn this study, we improved the structure of inclusion bodies (IBs) by optimizing the induction temperature, pH, concentrations of inducer, and metal ion additives. Statistically designed experimental analyses of three metal ion factors were performed using the Box-Behnken design. Induction temperature of 30°C, pH 7.0, and 0.1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) were selected, and the optimized levels for the factors predicted by the model comprised the following: Mg2+ (15.7 mM), Ca2+ (16.6 mM), and Mn2+ (3.0 mM). The optimized culture conditions improved the structure of the IBs, which was validated by scanning electron microscopy (SEM) and the increase of IBs solubility.ConclusionsAfter optimization, IB solubility and renatured mda-7/IL-24 increased by 51% and 84%, respectively. This study also provided a simple purification method of specific IB washing steps. Manipulating the fermentation parameters to optimize the refolding and purification process is likely to be widely applicable to other proteins.

Highly purified mussel adhesive protein to secure biosafety for in vivo applications.

BackgroundUnique adhesive and biocompatibility properties of mussel adhesive proteins (MAPs) are known for their great potential in many tissue engineering and biomedical applications. Previously, it was successfully demonstrated that redesigned hybrid type MAP, fp-151, mass-produced in Gram-negative bacterium Escherichia coli, could be utilized as a promising adhesive biomaterial. However, purification of recombinant fp-151 has been unsatisfactory due to its adhesive nature and polarity which make separation of contaminants (especially, lipopolysaccharide, a toxic Gram-negative cell membrane component) very difficult.ResultsIn the present work, we devised a high resolution purification approach to secure safety standards of recombinant fp-151 for the successful use in in vivo applications. Undesirable impurities were remarkably eliminated as going through sequential steps including treatment with multivalent ion and chelating agent for cell membrane washing, mechanical cell disruption, non-ionic surfactant treatment for isolated inclusion body washing, acid extraction of washed inclusion body, and ion exchange chromatography purification of acid extracted sample. Through various analyses, such as high performance liquid chromatographic purity assay, limulus amoebocyte lysate endotoxin assay, and in vitro mouse macrophage cell tests on inflammation, viability, cytotoxicity, and apoptosis, we confirmed the biological safety of bacterial-derived purified recombinant fp-151.ConclusionsThrough this purification design, recombinant fp-151 achieved 99.90% protein purity and 99.91% endotoxin reduction that nearly no inflammation response was observed in in vitro experiments. Thus, the highly purified recombinant MAP would be successfully used as a safety-secured in vivo bioadhesive for tissue engineering and biomedical applications.

Identification of protective immunogens from extracellular secretome of Edwardsiella tarda

Edwardsiella tarda is an opportunistic pathogen that causes a great loss in aquaculture. Identification of immune protective immunogens is a key step for development of subunit vaccines and control of the infectious diseases caused by the bacterium. This study aims to identify the protective antigens from extracellular secretory proteome of E. tarda. Out of 38 extracellular secretory proteins predicted by PSORTb, 20 genes were randomly cloned and their recombinant proteins were expressed in Escherichia coli BL21 and purified by either affinity chromatography or inclusion body washing. The purified recombinant proteins were used for investigation of immune protection in zebrafish model using active immunization approach. Half of them had significant immune protection compared with the control. Out of them, four, EseC, ETAE_2088, FlgD and ETAE_2130, showed approximately 60% relative percent survivals as a result of the highly protective antigens identified. Except for FlgD, the other three were first reported here. Moreover, the present study identified EseC and ETAE_2088 in bacterial extracellular fraction. These results indicate that secretory proteome is an interesting pool used for identification of immune protective antigens, and the four highly protective antigens identified provide useful candidates for development of subunit vaccines.

Expression, purification, and evaluation of recombinant LecA as a candidate for an amebic colitis vaccine

Entamoeba histolytica, which causes amebic colitis and liver abscess, is considered a major enteric pathogen in residents and travelers to developing countries where the disease is endemic. Interaction of this protozoan parasite with the intestine is mediated through the binding of the trophozoite stage to intestinal mucin and epithelium via a galactose and N-acetyl-d-galactosamine (Gal/GalNAc) lectin comprised of a disulfide linked heavy (ca. 180kDa) and light chain (ca. 35kDa) and a noncovalently bound intermediate subunit (ca. 150kDa). Our efforts to develop a vaccine against this pathogen have focused on an internal 578 amino acid fragment, designated LecA, located within the cysteine-rich region of the heavy chain subunit because: (i) it is a major target of adherence-blocking antibodies of seropositive individuals and (ii) vaccination with his-tagged LecA provides protection in animal models. We developed a purification process for preparing highly purified non-tagged LecA using a codon-optimized gene expressed in Escherichia coli. The process consisted of: (i) cell lysis, collection and washing of inclusion bodies; (ii) solubilization and refolding of denatured LecA; and (iii) a polishing gel filtration step. The purified fragment existed primarily as a random coil with β-sheet structure, contained low endotoxin and nucleic acid, was highly immunoreactive, and elicited antibodies that recognized native lectin and that inhibited in vitro adherence of trophozoites to CHO cells. Immunization of CBA mice with LecA resulted in significant protection against cecal colitis. Our procedure yields sufficient amounts of highly purified LecA for future studies on stability, immunogenicity, and protection with protein-adjuvant formulations.

Nasal administration of a novel recombinant human parathyroid hormone (1–34) analog for the treatment of osteoporosis of ovariectomized rats

Synthetic human parathyroid (1–34) (hPTH (1–34)) is known to have the full biological activity of the holohormone for osteoporosis. This study is about designing a novel analog of hPTH (1–34) which is more suitable for intranasal administration. We likewise evaluate effectiveness of the nasal drops against osteoroporosis. Through fusion expression of combining gene, cell disruption, inclusion body washing, ethanol fraction precipitation, acid hydrolysis, and CM-52 ion exchange column chromatography Pro-Pro-[Arg 11] hPTH (1–34)-Pro-Pro was designed and produced. Nasal drops of Pro-Pro-[Arg 11] hPTH (1–34)-Pro-Pro were prepared and administrated to ovariectomized rats. After 12 weeks of raising, Bone Material Densities (BMD) of vertebrae were examined by Dual Energy X-Ray Absorptiometry (DEXA). The average BMD of these groups treated with nasal drops of the peptide were 28.0%–47.2% (P < 0.01) higher than that of the group treated with normal saline (NS). The subchondral bone plates of the femoral heads were examined by scanning electron microscopy and a defined planar section was photographed. Percentage of the area of the cancellous bone was calculated. Percentages of the groups treated with nasal drops of the peptide increased; values were significantly different to that of the group treated with NS (P < 0.001) and were even equivalent to that of normal groups. These results show that nasal drops of Pro-Pro-[Arg 11] hPTH (1–34)-Pro-Pro are effective against osteoporosis.

Efficient preparation and PEGylation of recombinant human non-glycosylated erythropoietin expressed as inclusion body in E. coli

Recombinant human erythropoietin produced by mammalian cells contains about 40% carbohydrates which maintain its stability and long residence in body. However, mammalian derived Epo has low yields and high costs of production. In this article, a cost-effective strategy of producing non-glycosylated Epo from Escherichia coli and then PEGylating it to replace the role of sugar chains was investigated. Recombinant human non-glycosylated erythropoietin (rh-ngEpo) was overexpressed as inclusion body in E. coli. As the routine inclusion body washing step resulted in poor protein recovery and purity, a new process scheme of using strong ion-exchange chromatography to purify denatured rh-ngEpo from inclusion body before refolding was developed. The purity of the denatured rh-ngEpo was increased from 59% to over 90%. Rh-ngEpo was then refolded and subsequently purified by one step of weak cation-exchange chromatography to 98% pure. Final protein yield was 129 mg/l, a significant improvement from 49 mg/l obtained via the conventional practice. The in vitro bioactivity of purified rh-ngEpo was comparable with the CHO-expressed Epo and the formation of native secondary structure was also confirmed by CD spectra. Rh-ngEpo was then modified by a 20 kDa methoxy polyethylene glycol (PEG) succinimidyl carbonate. The monoPEGylated protein, which retained 68% bioactivity, had enhanced thermal stability and a remarkably prolonged circulating half-life in rats as compared with that of the unmodified protein. These studies demonstrated the feasibility of PEGylating rh-ngEpo as a promising way for the development of new Epo drugs.

Improving purification of recombinant human interferon γ expressed in Escherichia coli; effect of removal of impurity on the process yield

Process development and optimization studies were performed in order to improve the purification process of (rhIFN-γ). The objective was to generate material with higher purity and quantity. An in-process control screening was developed to obtain the optimal condition for column chromatographic purification by measuring LPS, nucleic acids, rhIFN- γ, monomer and its covalent dimers. A new resin screening method was applied to select optimal resin for each of the chromatographic columns. The resulting process used Butyl and Q-Sepharose, refolding and SP-Sepharose for purification of IFN-γ. Effects of different process conditions such as cell lysis, removal of impurity and oxygen concentration were evaluated. Removal of impurities was evaluated by washing of inclusion bodies with 1% Triton X-100 and 3 M urea and different chromatography steps. The results reveal that Triton removed about 43% of the LPS but urea had no effect on removal of nucleic acids and LPS. Further analysis show that removal of impurities by column chromatography decreases aggregation and increases the process yield. Oxygen concentration was identified as parameter that could have a significant impact on covalent dimers formation, as an unacceptable pharmaceutical form of rhIFN-γ. On the basis of small-scale studies, optimum operating conditions were chosen and the purification process was successfully scaled-up to a pilot scale process with step yield and product quality that were better than previous reports.

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.

Optimization of the primary recovery of human interferon α2b from Escherichia coli inclusion bodies

The human interferon α2b (hu-IFNα2b) gene was cloned in Escherichia coli JM109(DE3) and the recombinant protein was expressed as cytoplasmic inclusion bodies (IB). The present work discusses the recovery of hu-IFNα2b IB from the E. coli cells. An optimized protocol is proposed based on the sequential evaluation of recovery steps and parameters: (i) cell disruption, (ii) IB recovery and separation from cell debris, (iii) IB washing, and (iv) IB solubilization. Parameters such as hu-IFNα2b purity and recovery yield were measured after each step. The optimized recovery protocol yielded 60% of hu-IFNα2b with a purity of up to 80%. The protein was renatured at high concentration after recovery and it was found to display biological activity.

Effect of operating variables on the yield of recombinant trypsinogen for a pulse-fed dilution-refolding reactor.

The inclusion body process route for manufacturing proteins offers distinct process advantages in terms of expression levels and the ease of initial inclusion body recovery. The efficiency of the refolding unit operation, however, does determine the overall economic feasibility of a process. Dilution refolding is the simplest and most extensively used refolding operation, although significant yield losses often occur due mainly to aggregation. Operating variables may have a significant effect on the degree of aggregation, but a systematic study has not been reported. This study investigates the effect of operating variables on the dilution refolding of solubilized r-trypsinogen inclusion bodies in a pulse-fed stirred reactor. Variables investigated were inclusion body washing, stirring speed, feed rate, concentration of solubilized r-trypsinogen, and concentration of urea during solubilization of the inclusion bodies. Additionally, the effect of baffles in the reactor was investigated. The yield of renatured r-trypsinogen varied between 12 +/- 0.2% and 21 +/- 1.0% depending on the specific combination of operating variables employed. It is clear that a suboptimal operating strategy can significantly reduce protein yield. In particular, we note that an increased intensity of mixing adversely affected yield in contrast to previous reports indicating that enhanced dispersion increases yield. We conclude that yield is determined not only by the efficiency of dispersion, but also by the local chemical environment of the protein as it folds, and the rate of change of this environment. This will be controlled by micromixing effects, and hence the intensity of agitation, in a complex manner requiring further characterization.

Recombinant flounder growth hormone from Escherichia coli: overexpression, efficient recovery, and growth-promoting effect on juvenile flounder by oral administration

An efficient production method for recombinant flounder growth hormone (r-fGH) from Escherichia coli was developed and the biological activity of purified r-fGH was examined using juvenile flounder. The use of bicistronic construction in the expression plasmid resulted in the production of over 40% of the E. coli cellular protein as r-fGH. The r-fGH was recovered from cell lysates following inclusion body washing, solubilization and refolding in sodium dodecylsulfate (SDS) solution, and removal of contaminated proteins with secondary butanol treatment. The SDS content in purified r-fGH solution was adjusted to appropriate levels by diafiltration. More than 47% of the r-fGH was recovered from the E. coli cell lysates and the purity of recovered r-fGH was 98%. The oral administration of purified r-fGH to juvenile flounder, once a week for 4 weeks at a dosage of 40 μg r-fGH g −1 fish body weight, resulted in significant increases both in weight and length. These results of overexpression, simple purification with high recovery yield and purity, and good growth-promoting activity of the r-fGH suggest that the production scheme described in this study is useful for the potential application of r-fGH in fish farming.

The effect of denaturants on the crossflow membrane filtration of Escherichia coli lysates containing inclusion bodies

Recovery of inclusion bodies from Escherichia coli cell lysates often includes a washing step to remove associated contaminants that may otherwise interfere with refolding of the solubilized protein. The effects of denaturants typically used in the washing step on the cell lysate viscosity and membrane filtration performance were examined. Guanidine HCl alone and in combination with the nonionic detergent, Triton X-100, had the greatest effect on the cell lysate viscosity, the dead-end filtration rate and the performance of the crossflow membrane filters. Triton X-100 reduced the dead-end filtration rate, but had less of an effect than guanidine HCl on permeate flux during crossflow membrane filtration. Urea had minimal effect on either the dead-end filtration rate or the permeate flux and protein transmission during crossflow membrane filtration. The use of low concentrations of urea along with a PVDF membrane allowed for inclusion body washing without detrimental changes in the permeate flux and protein transmission during crossflow membrane filtration.

Purification and renaturation of recombinant human lymphotoxin (tumour necrosis factor beta) expressed in Escherichia coli as inclusion bodies

High level expression of recombinant human tumour necrosis factor beta (rhTNF-beta) in Escherichia coli results in the formation of two portions of protein, namely soluble active protein and insoluble protein which is inactive and aggregates in the form of inclusion bodies (IBs). In this study, a procedure for purification and renaturation of rhTNF-beta from inclusion bodies has been designed and verified experimentally with a product purity of more than 90% and a recovery of about 30%. The procedure includes washing of IBs with specific wash buffer (Triton X-100/EDTA/lysozyme/PMSF), their solubilization with 8 mol dm-3 alkaline urea, purification with ion-exchange columns, refolding with renaturation buffer and finally concentration and desalination with an ultrafiltration membrane. The characteristics of the renatured protein were identical with those of purified protein from the soluble fraction as demonstrated by (1) SDS-PAGE, (2) cytotoxic activity on mouse L929 cells, (3) N-terminal amino acid sequence, and (4) gel filtration chromatography.