Purification Of Recombinant Proteins Research Articles

Pichia pastoris: A highly successful expression system for optimal synthesis of heterologous proteins.

One of the most important branches of genetic engineering is the expression of recombinant proteins using biological expression systems. Nowadays, different expression systems are used for the production of recombinant proteins including bacteria, yeasts, molds, mammals, plants, and insects. Yeast expression systems such as Saccharomyces cerevisiae (S. cerevisiae) and Pichia pastoris (P. pastoris) are more popular. P. pastoris expression system is one of the most popular and standard tools for the production of recombinant protein in molecular biology. Overall, the benefits of protein production by P. pastoris system include appropriate folding (in the endoplasmic reticulum) and secretion (by Kex2 as signal peptidase) of recombinant proteins to the external environment of the cell. Moreover, in the P. pastoris expression system due to its limited production of endogenous secretory proteins, the purification of recombinant protein is easy. It is also considered a unique host for the expression of subunit vaccines which could significantly affect the growing market of medical biotechnology. Although P. pastoris expression systems are impressive and easy to use with well‐defined process protocols, some degree of process optimization is required to achieve maximum production of the target proteins. Methanol and sorbitol concentration, Mut forms, temperature and incubation time have to be adjusted to obtain optimal conditions, which might vary among different strains and externally expressed protein. Eventually, optimal conditions for the production of a recombinant protein in P. pastoris expression system differ according to the target protein.

AMPK Complex Activation Promotes Sarcolemmal Repair in Dysferlinopathy

Mutations in dysferlin are responsible for a group of progressive, recessively inherited muscular dystrophies known as dysferlinopathies. Using recombinant proteins and affinity purification methods combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS), we found that AMP-activated protein kinase (AMPK)γ1 was bound to a region of dysferlin located between the third and fourth C2 domains. Using ex vivo laser injury experiments, we demonstrated that the AMPK complex was vital for the sarcolemmal damage repair of skeletal muscle fibers. Injury-induced AMPK complex accumulation was dependent on the presence of Ca2+, and the rate of accumulation was regulated by dysferlin. Furthermore, it was found that the phosphorylation of AMPKα was essential for plasma membrane repair, and treatment with an AMPK activator rescued the membrane-repair impairment observed in immortalized human myotubes with reduced expression of dysferlin and dysferlin-null mouse fibers. Finally, it was determined that treatment with the AMPK activator metformin improved the muscle phenotype in zebrafish and mouse models of dysferlin deficiency. These findings indicate that the AMPK complex is essential for plasma membrane repair and is a potential therapeutic target for dysferlinopathy.

Exploring the Mesenchymal Stem Cell Secretome for Corneal Endothelial Proliferation.

Ex vivo grown human corneal endothelial cells (HCEnC) are a new emerging treatment option to treat visually impaired patients aimed at alleviating the current global donor shortage. Expanding HCEnC is still challenging, and obtaining cells in sufficient quantities is a limiting factor. It is already known that conditioned medium obtained from bone marrow mesenchymal stem cells can stimulate the proliferation of endothelial cells. The aim of this study was to take this work a step further to identify some of the underlying factors responsible. We confirmed the stimulatory effect of the mesenchymal stem cell secretome seen previously and separated the exosomes from the soluble proteins using size exclusion chromatography. We demonstrated the presence of exosomes and soluble proteins in the early and late fractions, respectively, with transmission electron microscopy and protein assays. Proliferation studies demonstrated that growth stimulation could be reproduced with the later protein-rich fractions but not with the exosome-rich fraction. Antibody assays revealed the presence of the secreted proteins EGF, IGFBP2, and IGFBP6 in protein-high fractions, but the growth enhancement was not seen with purified protein formulations. In conclusion, we confirmed the stimulatory effect of stem cell-conditioned medium and have determined that the effect was attributable to the proteins rather than to the exosomes. We were not able to reproduce the growth stimulation, however, with the pure recombinant protein candidates tested. Specific identification of the underlying proteins using proteomics could render a bioactive protein that can be used for ex vivo expansion of cells or as an in vivo drug to treat early corneal endothelial damage.

Chitosan-coated magnetic iron oxide nanoparticles for DNA and rhEGF separation

The obtention and purification of DNA and recombinant proteins are critical steps in the biotech industries. In this research, the use of chitosan-coated magnetic iron oxide nanoparticles as magnetic nano-adsorbent was investigated. Iron oxide nanoparticles were obtained through a simple coprecipitation method. The spinel structure of the nanoparticles was confirmed by X-Ray diffraction analysis. The particle size before (16 nm) and after chitosan coating (14 nm) was measured using scanning electron microscopy. Infrared spectroscopy and thermogravimetric analysis measurements confirmed the presence of chitosan on the surface of magnetic nanoparticles coated in a percentage of 11.24%. The Redlich-Peterson isotherm yielded the best fit for the DNA experimental adsorption capacity and a maximum of 98 mg/g was obtained. The structural integrity of DNA, after the elution process, was confirmed by agarose gel electrophoresis. An adsorption capacity of 440 mg/g for rhEGF was found and the Langmuir-Freundlich isotherm showed the best fit for the experimental results. Finally, SDS-PAGE and Western blot assays confirmed that the adsorption/desorption process did not affect the rhEGF identity, thereby, suggesting that the biological activity was preserved.

Expression, Purification and Docking Studies on IMe-AGAP, the First Antitumor-analgesic Like Peptide from Iranian Scorpion Mesobuthus eupeus.

Scorpion venom contains different toxins with multiple biological functions. IMe-AGAP is the first Analgesic-Antitumor like Peptide (AGAP) isolated from Iranian scorpion Mesobuthus eupeus. This peptide is similar to AGAP toxin with high analgesic activity, extracted from Chinese scorpion and inhibits NaV1.8 and NaV1.9 voltage-gated sodium channels involved in the pain pathway. In this study, IMe-AGAP was cloned in a prokaryotic expression vector; expression of toxin in Escherichia coli (E. coli) was assayed and then purified. In in-silico studies, peptide sequence was compared with other scorpion analgesic toxins. The structures of IMe-AGAP and sodium channels were modeled using homology modeling. Structural evaluation and stereo-chemical analysis of modeled structures were performed using RAMPAGE web server Ramachandran plots. Hex Server was used to investigate the interactions between IMe-AGAP and S3-S4 and also S5-S6 segments of NaV1.8 and NaV1.9. Binding energies calculation was used for evaluation of protein docking. Soluble expression of IMe-AGAP in bacteria was investigated by SDS-PAGE analysis. Pure recombinant protein was obtained by Ni-NTA affinity chromatography. The results of three-dimensional structure prediction showed βαββ topology for the toxin that is similar to the conserved structure of α-toxins. Comparison analysis between IMe-AGAP and AGAP toxins exhibited high similarity in homology modeling. Docking analysis demonstrated that IMe-AGAP can interact with NaV1.8 and NaV1.9 domains involved in pain. According to the results of homology studies and docking, IMe-AGAP might be a novel potential drug for pain treatment.

Expression and Purification of Full-Length and Domain-Fragment Recombinant Pentraxin 3 (PTX3) Proteins from Mammalian and Bacterial Cells.

Although cell-based protein expression systems enable us a certain amount of protein suitable for subsequent biological experiments to be obtained, aggregates of the protein of interest are sometimes encountered during the purification procedure. Pentraxin 3 (PTX3), a member of the pentraxin family that is classified as a carbohydrate-binding protein based on its structure, comprises one of the humoral arms of the pattern recognition receptors that play an important role in the innate immune response. PTX3 comprises two domains; an N-terminal domain and a C-terminal domain. The C-terminal domain containing pentraxin signature has similar biological functions as other pentraxins such as C-reactive protein (CRP) and serum amyloid-P component (SAP). On the other side, the N-terminal domain is specific to PTX3. A supply of the PTX3 protein in full length or partial fragments is thus essential for the elucidation of its biological functions. Here we describe the expression and purification of recombinant PTX3. Anarginine-containing bufferis essential for the elution of bacterially expressed PTX3 N-terminal domain to minimize aggregation. This method allows high-yield purification of full-length or domain-fragment recombinant PTX3 proteins for biological study.

High pressure homogenization is a key unit operation in inclusion body processing

Recombinant protein production in E. coli often leads to the formation of inclusion bodies (IBs). Although downstream processing of IBs has the reputation of being a great hurdle, advantages of IBs can be substantial. Highly pure recombinant protein with the possibility of correctly folded structures and an easy separation from cell matter are decisive factors that make IB processes so interesting. Product yield, purity and biological activity of the refolded protein are the responses to evaluate an IB process. The objective of this case study was to develop a refolding process in an integrated manner. The effects of the unit operations 1) homogenization, 2) IB wash and 3) IB solubilisation as well as their interdependencies were analyzed. We revealed interesting factor interactions between homogenization and IB wash, as well as homogenization and solubilisation, which would be overlooked if the single unit operations were investigated individually. Furthermore, we found that homogenization was a key unit operation for IB processing. By changing the conditions during homogenization only, the product yield, purity and biological activity of the refolded product was affected 2-fold, 1.2-fold and 2.5-fold, respectively.

A systematic and methodical approach for the efficient purification of recombinant protein from silkworm larval hemolymph

The silkworm, Bombyx mori, is a promising expression system for the production of recombinant proteins, but the purification of these proteins is not easy because of the large amount of host proteins present. To investigate purity, recovery and scale-up ability of the purification of recombinant proteins expressed in silkworm larval hemolymph without any affinity tags, we used mCherry, a red fluorescence protein, as a model. The host cell proteins could be greatly reduced using a three-step chromatography protocol consisting of hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC) and heparin chromatography after heat pretreatment. The thermal treatment had the greatest impact on the removal of host cell extracellular proteins and increasing purity. There were still some minor traces of host cell proteins in the purified sample, which showed that the purification of recombinant proteins from the silkworm hemolymph was still challenging. The proposed protocol and affinity tag purification reduced the overall protein content by 99.84% and 99.95%, respectively, while the amount of DNA was reduced by 98.41% and 99.53%, respectively. Purities of our proposed protocol based on SDS-PAGE and capillary electrophoresis (CE) analyses were 85.45% and 43.60%, respectively, while those of Strep-tag affinity purification were 100% or 63.69%, respectively. Using densitometry, the overall recovery was calculated was 5.78%, which was higher than 4.09% using Strep-tag affinity purification. This proposed protocol, mainly based on thermal treatment, HIC, SEC and HiTrap Heparin HP column chromatography, is applicable to an upscalable purification for the silkworm expression system without employing affinity tag chromatography process.

Bacterial sugar-binding protein as a one-step affinity purification tag on dextran-containing resins

Marinobacter hydrocarbonoclasticus is an oil-eating bacterium that possesses a large adhesion protein (MhLap) with the potential to bind extracellular ligands. One of these ligand-binding modules is the ~20-kDa PA14 domain (MhPA14) that has affinity for glucose-based carbohydrates. Previous studies showed this sugar-binding domain is retained on dextran-based size-exclusion resins during chromatography, requiring the introduction of glucose or EDTA to remove the protein from the column. Given the ready availability of such size-exclusion resins in biochemistry laboratories, this study explores the use of MhPA14 as an affinity tag for recombinant protein purification. Two different fusion proteins were tested: 1) Green fluorescent protein (GFP) linked to the N-terminus of the MhPA14 tag; and 2) the ice-binding domain from the Marinomonas primoryensis ice-binding protein (MpIBD) linked to the MhPA14 C-terminus by a TEV cut site. The GFP_MhPA14 fusion visibly bound to Superdex, Sephadex, and Sephacryl resins, but did not bind to Sepharose. Using Superdex resin, dextran-affinity purification proved to be an effective one-step purification strategy for both proteins, superior to even nickel-affinity chromatography. Dextran-affinity chromatography was also the most effective method of separating the MhPA14 tag from MpIBD following TEV proteolysis, as compared to both nickel-affinity and ice-affinity methods. These results indicate that MhPA14 has potential for widespread use in recombinant protein purification.

Two Different Missense C1S Mutations, Associated to Periodontal Ehlers-Danlos Syndrome, Lead to Identical Molecular Outcomes.

Ehlers-Danlos syndromes (EDS) are clinically and genetically heterogeneous disorders characterized by soft connective tissue alteration like joint hypermobility and skin hyper-extensibility. We previously identified heterozygous missense mutations in the C1R and C1S genes, coding for the complement C1 proteases, in patients affected by periodontal EDS, a specific EDS subtype hallmarked by early severe periodontitis leading to premature loss of teeth and connective tissue alterations. Up to now, there is no clear molecular link relating the nominal role of the C1r and C1s proteases, which is to activate the classical complement pathway, to these heterogeneous symptoms of periodontal EDS syndrome. We aim therefore to elucidate the functional effect of these mutations, at the molecular and enzymatic levels. To explore the molecular consequences, a set of cell transfection experiments, recombinant protein purification, mass spectroscopy and N-terminal analyses have been performed. Focusing on the results obtained on two different C1S variants, namely p.Val316del and p.Cys294Arg, we show that HEK293-F cells stably transfected with the corresponding C1s variant plasmids, unexpectedly, do not secrete the full-length mutated C1s, but only a truncated Fg40 fragment of 40 kDa, produced at very low levels. Detailed analyses of the Fg40 fragments purified for the two C1s variants show that they are identical, which was also unexpected. This suggests that local misfolding of the CCP1 module containing the patient mutation exposes a novel cleavage site, between Lys353 and Cys354, which is not normally accessible. The mutation-induced Fg40 fragment contains the intact C-terminal serine protease domain but not the N-terminal domain mediating C1s interaction with the other C1 subunits, C1r, and C1q. Thus, Fg40 enzymatic activity escapes the normal physiological control of C1s activity within C1, potentially providing a loss-of-control. Comparative enzymatic analyses show that Fg40 retains the native esterolytic activity of C1s, as well as its cleavage efficiency toward the ancillary alarmin HMGB1 substrate, for example, whereas the nominal complement C4 activation cleavage is impaired. These new results open the way to further molecular explorations possibly involving subsidiary C1s targets.

Variations of binding, washing, and concentration of imidazole on purification of recombinant Fim-C Protein Salmonella typhi with Ni-NTA Resin

Typhoid fever is an endemic disease in Indonesia. Prevention of typhoid fever can be done by administering vaccines. It is known that one of the raw materials for vaccines is a recombinant protein. This study aims to obtain information on the optimum conditions for purification of Salmonella typhi Fim-C recombinant protein with Ni-NTA resin as vaccine raw material. The three main stages of the purification process in this study were binding, washing, and elution of S. typhi Fim-C recombinant proteins. The binding and washing variations of recombinant proteins were carried out twice, four times and six times, while the elution process was carried out at imidazole concentrations of 200 mM, 250 mM, and 300 mM. Purification with a binding process four and six times gave almost the same intensity of S. typhi Fim-C protein bands. Whereas protein was elution at an imidazole concentration of 300 mM showed higher band instability. The results of characterization using SDS-PAGE and analysis using software ImageJ gel analysis showed that the longer the incubation time and the repetition of the binding process, the more protein bound to the resin. Furthermore, the more washing processes are obtained the purer proteins. Based on the data obtained it can be concluded that the purification of the S. typhi Fim-C recombinant protein was optimum at a four-time binding process, six times washing and a 300 mM imidazole concentration. These results are expected to be the basis for recombinant protein refining on a pilot scale and industry scale in better vaccine preparation.

Direct Cloning Method for Expression of Recombinant Proteins with an Inositol Hexakisphosphate Inducible Self-Cleaving Tag.

Protein purification is the most basic and critical step for protein biophysical and biochemical studies to understand its function and structure. Various fusion tags and proteases have been developed and assembled in expression and purification system. However, it is one of the fields that continues to innovateto develop improved systems that are more efficient, simpler, and less expensive. An efficient self-cleavage C-terminal fusion system was developed using the inositol hexakisphosphate-inducible Vibrio cholerae MARTXVc toxin cysteine protease domain (CPD). CPD fusion proteins are expressed from the T7 promoter and purified using a 6xHis-tag with immobilized-metal affinity chromatography. The C-terminal CPD-tag is removed by self-cleavage at the final purification stage. Here, we describe an efficient cloning method using Gibson assembly, followed by expression and purification of tagless recombinant proteins of interest using CPD self-cleavage.

Modeling and simulation of anion exchange chromatography for purification of proteins in complex mixtures

Ion exchange chromatography is extensively used in the purification of biological compounds. Reliable mathematical models describing this chromatographic technique are available and can be used to improve the performance of this separation step. However, the use of synthetic mixtures for model development hampers the application of this approach with real cell extracts processed in downstream operations. This work presents an original approach for handling non-synthetic genuine mixtures of proteins, which was applied in the purification of an untagged recombinant pneumococcal surface protein A (PspA4Pro). First, evaluation was made of the efficiency of steric mass action (SMA) and modified Langmuir isotherms, which were separately used together with the equilibrium dispersive model (EDM). The data used for parameter estimation and model validation were obtained from anion exchange chromatography runs (employing Q-Sepharose FF), applied to real cell extracts produced by different cultivation strategies. Simulations showed that the models were able to describe the complex mixtures of unknown proteins. Next, the EDM and SMA approaches were used to separately describe the profile of PspA4Pro and the pool of protein impurities eluted together. The simulations showed that PspA4Pro tended to elute at the beginning of the peak, enabling the establishment of an alternative elution schedule that provided a 34% increase in the purity achieved using the anion exchange chromatography.

A bacterial assay for rapid screening of IAA catabolic enzymes

BackgroundPlants rely on concentration gradients of the native auxin, indole-3-acetic acid (IAA), to modulate plant growth and development. Both metabolic and transport processes participate in the dynamic regulation of IAA homeostasis. Free IAA levels can be reduced by inactivation mechanisms, such as conjugation and degradation. IAA can be conjugated via ester linkage to glucose, or via amide linkage to amino acids, and degraded via oxidation. Members of the UDP glucosyl transferase (UGT) family catalyze the conversion of IAA to indole-3-acetyl-1-glucosyl ester (IAGlc); by contrast, IAA is irreversibly converted to indole-3-acetyl-l-aspartic acid (IAAsp) and indole-3-acetyl glutamic acid (IAGlu) by Group II of the GRETCHEN HAGEN3 (GH3) family of acyl amido synthetases. Dioxygenase for auxin oxidation (DAO) irreversibly oxidizes IAA to oxindole-3-acetic acid (oxIAA) and, in turn, oxIAA can be further glucosylated to oxindole-3-acetyl-1-glucosyl ester (oxIAGlc) by UGTs. These metabolic pathways have been identified based on mutant analyses, in vitro activity measurements, and in planta feeding assays. In vitro assays for studying protein activity are based on producing Arabidopsis enzymes in a recombinant form in bacteria or yeast followed by recombinant protein purification. However, the need to extract and purify the recombinant proteins represents a major obstacle when performing in vitro assays.ResultsIn this work we report a rapid, reproducible and cheap method to screen the enzymatic activity of recombinant proteins that are known to inactivate IAA. The enzymatic reactions are carried out directly in bacteria that produce the recombinant protein. The enzymatic products can be measured by direct injection of a small supernatant fraction from the bacterial culture on ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem spectrometry (UHPLC–ESI-MS/MS). Experimental procedures were optimized for testing the activity of different classes of IAA-modifying enzymes without the need to purify recombinant protein.ConclusionsThis new method represents an alternative to existing in vitro assays. It can be applied to the analysis of IAA metabolites that are produced upon supplementation of substrate to engineered bacterial cultures and can be used for a rapid screening of orthologous candidate genes from non-model species.

High yield recombinant expression and purification of oncogenic NSD1, NSD2, and NSD3 with human influenza hemagglutinin tag

The nuclear receptor-binding SET Domain (NSD) family consists of NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L1 histone methyltransferases that are crucial for chromatin remodeling. NSDs are implicated in developmental disorders such as Wolf-Hirschhorn and Sotos syndromes as well as various cancers including t(4; 14)(p16; q32) myeloma, an incurable cancer in plasma cells. NSDs have been the target of intensive study to understand their biological functions more fully and inform anti-cancer drug design. Recombinant protein expression and purification of human NSDs using an E. coli expression system are notoriously challenging, but the production of pure, stable, and active NSDs is essential for further studies. To overcome production challenges, we propose a cost-efficient approach optimized to produce a high yield of NSDs using a modified E. coli expression system. We found that tagging the NSDs with a human influenza hemagglutinin (HA) tag greatly improved the quality of the recombinant NSDs, resulting in more than 95% pure, stable, and active NSD-HAs, with an increase in production yield up to 22.4-fold and up to 6.25 mg/L from LB E. coli culture, and without further purification such as ion-exchange or size-exclusion chromatography.

Spray-Dried Hierarchical Aggregates of Iron Oxide Nanoparticles and Their Functionalization for Downstream Processing in Biotechnology.

In this work, the structuring of iron oxide nanoparticles via spray-drying (SD) of aqueous suspensions is investigated, leading to micrometer-sized aggregates with saturation magnetization comparable to that of the individual nanoparticles. Interestingly, the superparamagnetic behavior is retained despite the multicore structure. Modification of the aggregates via the addition of silica nanoparticles to the suspension allows for control of the resulting magnetization by adjusting the iron oxide content. Moreover, the morphology of the produced aggregates is gradually shifted from irregular inflated-like shapes in case of pure iron oxide aggregates to reach spherical structures when bringing the silica content to only 20%. The aggregates with different magnetization can be effectively separated in a simple column with an attached permanent magnet. Functionalization of pure iron oxide aggregates with a previously coupled ligand holding a nitrilotriacetic acid (NTA)-like moiety and subsequent loading with Ni2+ ions leads to the ability to bind 6-histidine (His6)-tagged target proteins via chelation complexes for magnetic separation. The application of the presented system for the purification of recombinant protein A in multiple cycles is shown. The recyclability of the separation system in combination with the high degree of magnetic separation is promising for future applications in the field of preparative in situ protein purification.

ADAMTS Proteins: Concepts, Challenges, and Prospects.

The ADAMTS superfamily comprises secreted metalloproteases (ADAMTS proteases) as well as structurally related secreted glycoproteins that lack catalytic activity (ADAMTS-like proteins). Members of both families participate in diverse morphogenetic processes during embryonic development, and connective tissue maintenance and hemostasis in the adult. Several ADAMTS proteins are heavily implicated in genetic and acquired human and animal disorders. Despite these indicators of a profound biological and medical importance, detailed knowledge about their molecular structures, substrates, biological pathways, and biochemical mechanisms is significantly limited by unique intrinsic characteristics, which have led to several technical challenges. As a group, they are larger, more heavily modified, and harder to purify than other secreted proteases. In addition, idiosyncratic aspects of individual members are deserving of further investigation but can complicate their analysis. Here, some of the key concepts, challenges, and prospects in ADAMTS research are discussed in the context of the knowledge accumulated over the past two decades. Individual chapters in this volume of Methods in Molecular Biology provide practical solutions for surmounting these challenges. Since the biology of a protease is actually the biology of its substrates, there is considerable emphasis on purification of recombinant ADAMTS proteins, identification of their substrates and assays for their proteolytic activity.

Characterization of IgE-binding proteins in the salivary glands of Simulium nigrogilvum (Diptera: Simuliidae).

Simulium dermatitis is an IgE-mediated skin reaction in animals and humans caused by the bites of black flies. Although Simulium nigrogilvum has been incriminated as the main human-biting black fly species in Thailand, information on its salivary allergens is lacking. Salivary gland extract of S. nigrogilvum females was subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis, and the separated components were applied onto nitrocellulose membranes for immunoblotting, which was performed by probing the protein blots with sera from 17 individuals who were allergic to the bites of S. nigrogilvum. IgE-reactive protein bands were characterized further by liquid chromatography-mass spectrometry (LC-MS/MS) analysis. Nine protein bands (79, 42, 32, 25, 24, 22, 15, 13, and 11kDa) were recognized in the serum of the subjects. Four of the nine protein bands (32, 24, 15, and 11kDa) showed IgE reactivity in all (100%) of the tested sera, and they were identified as salivary secreted antigen 5-related protein, salivary serine protease, erythema protein, and hypothetical secreted protein, respectively. Three other proteins, salivary serine protease (25kDa), salivary D7 secreted protein (22kDa), and hypothetical protein (13kDa), reacted with > 50% of the sera. The relevance of the identified protein bands as allergens needs to be confirmed by using pure recombinant proteins, either in the in vivo skin prick test or in vitro detection of the specific IgE in the serum samples of allergic subjects. This will be useful for the rational design of component-resolved diagnosis and allergen immunotherapy for the allergy mediated by the bites of black flies.

Expression and purification of recombinant coat protein of sugarcane mosaic virus from Indonesian isolate as an antigen for antibody production

Sugarcane mosaic virus (SCMV, genus Potyvirus, family Potyviridae) is a prominent pathogen of sugarcane (Saccharum sp. hybrids). It can cause losses in susceptible varieties, in crop as well as sugar production, economically. Although it has been studied in major sugar-producing countries, research on the definement of SCMV from Indonesian isolates based on molecular study has been very limited. This study aimed to obtain a proper recombinant antigens emanating from coat protein of SCMV from Indonesian isolate in order to produce polyclonal antibodies that cann be used for immunodiagnosis assays in a subsequent study. A gene-encoding coat protein of SCMV (CP-SCMV) was amplified using RT-PCR and cloned into vector pJET1.2. The cDNA was inserted into 6X His-tag expression plasmid of pET28a(+) and over-expressed in Escherichia coli BL21(DE3) to produce a recombinant protein. The highest expression was found in 0.1M IPTG induction media for 5 h at 37oC. SDS-PAGE analysis clarified that the recombinant CP-SCMV remained as an insoluble fraction. Purifications was carried out by the affinity Ni-NTA resin, followed by electroelution to obtain a highly purified protein. To meet the quality requirements of a proper antigen, the highly purified protein was concentrated. A molecular weight of the rCP-SCMV (approximately 40 kDa) was clearly observed by 10% SDS-PAGE at the concentration of 16.184 mg/mL.

Detection and quantification of transition metal leaching in metal affinity chromatography with hydroxynaphthol blue

The widespread use of immobilized metal-affinity chromatography (IMAC) for fast and efficient purification of recombinant proteins has brought potentially toxic transition elements into common laboratory usage. However, there are few studies on the leaching of metal from the affinity resin, such as nickel-nitrilotriacetic acid (Ni-NTA), with possible deleterious impact on the biological activity. This is of particular importance when reducing or chelating eluants stronger than imidazole are used. We present a detailed study of hydroxynaphthol blue (HNB) as an indicator of several divalent metal cations, but with emphasis on Ni2+, clarifying and correcting many errors and ambiguities in the older literature on this dye compound. The assay is simple and sensitive and many metals, notably Ni2+, Zn2+, Cu2+, Pb2+, Fe2+, Co2+, and Al3+, can be readily detected and quantified at concentrations down to 15–50 nM (1–5 ppb) at neutral pH and in most commonly used buffers using spectroscopic equipment available in typical biochemistry research labs. Using this method, we show that significant amounts of Ni2+ (up to 20 mM) are co-purified with a target protein (cytochrome bc1 complex) when histidine is used to elute from Ni-NTA resin.