His-tagged Recombinant Protein Research Articles

Response to oxidative stress involves a novel peroxiredoxin gene in the unicellular cyanobacterium Synechocystis sp. PCC 6803.

Exposure to methyl viologen in the presence of light facilitates the production of superoxide that gives severe damage on photosynthetic apparatus as well as many cellular processes in cyanobacteria and plants. The effects of methyl viologen on global gene expression of a unicellular cyanobacterium Synechocystis sp. strain PCC 6803 were determined by DNA microarray. The ORFs sll1621, slr1738, slr0074, slr0075, and slr0589 were significantly induced by treatment of methyl viologen for 15 min commonly under conditions of normal and high light. One of these genes, slr1738, which encodes a ferric uptake repressor (Fur)-type transcriptional regulator, is located divergently next to another induced gene, sll1621, in the genome. We deleted slr1738, and compared the global gene expression patterns of this mutant to that of wild type under non-stressed conditions. It was found that sll1621 was derepressed to the greatest extent, while many other genes including slr0589 but not slr0074 or slr0075 were derepressed to lesser extent in the mutant. Genetic disruption of sll1621, which encodes a putative type 2 peroxiredoxin, indicates that it is essential for aerobic phototrophic growth in both liquid and solid media in high light and on solid medium even in low light. Slr1738 was prepared as a His-tagged recombinant protein and shown to specifically bind to the intergenic region between sll1621 and slr1738. The binding was enhanced by dithiothreitol and abolished by hydrogen peroxide. We concluded that the Fur homolog, Slr1738, plays a regulatory role in the induction of a potent antioxidant gene, sll1621, in response to oxidative stress.

Arabidopsis MBD proteins show different binding specificities and nuclear localization.

Recent results in animals and plants have shown a strong link between DNA methylation, chromatin structure and epigenetic control. In plants DNA methylation affects both symmetric and asymmetric cytosines by means of different DNA-methyltransferases. In vertebrates these modifications are interpreted by a group of proteins (methylated DNA-binding domain proteins, MBDs) able to specifically bind methylated CpG. In plants several genes sharing structural homology to mammalian MBD have been identified in Arabidopsis and maize, but their characterization is still to be completed. Here we present the characterization of six different MBDs from Arabidopsis. As judged by semi-quantitative RT-PCR, their expression proved to be differentially modulated in different organs. All the corresponding polypeptides, expressed in Escherichia coli as His-tagged recombinant proteins, have been functionally tested on gel shift experiments but only two of them (namely MBD5, 6) were able to specifically bind methylated CpG oligonucleotides. A third protein, AtMBD11, showed a strong affinity for DNA independently from the level of methylation. Moreover we were able to differentiate MBD5 and 6, despite their high homology, for their ability to recognize methylated asymmetrical sites. The binding specificity of these three AtMBD proteins was tested not only on arbitrarily chosen probes but also on the Arabidopsis E2F recognition sequence containing a single CpG site. Protoplasts transient expression experiments of GFP-fusion proteins showed for AtMBD5 and AtMBD6 a heterochromatic localization which was affected by 5-azacytidine treatment. These data demonstrate that AtMBD5 and AtMBD6 bind methylated DNA in vitro and in vivo with different specificity and might therefore have different roles in methylation-mediated transcriptional silencing.

PPE Protein (Rv3873) from DNA Segment RD1 ofMycobacterium tuberculosis: Strong Recognition of Both Specific T-Cell Epitopes and Epitopes Conserved within the PPE Family

Proteins encoded by DNA segment RD1 of Mycobacterium tuberculosis have recently been demonstrated to play important roles in bacterial virulence, vaccine development, and diagnostic reagent design. Previously, we characterized two immunodominant T-cell antigens, the early secreted antigen target (ESAT-6) and the 10-kDa culture filtrate protein (CFP10), which are encoded by the esx-lhp operon in this region. In the present study we characterized a third putative open reading frame in this region, rv3873, which encodes a PPE protein. We found that the rv3873 gene is expressed in M. tuberculosis H37Rv and that the native protein, Rv3873, is predominantly associated with the mycobacterial cell or wall. When tested as a His-tagged recombinant protein, Rv3873 stimulated high levels of gamma interferon secretion in peripheral blood mononuclear cells isolated from tuberculosis (TB) patients, as well as from healthy tuberculin purified protein derivative-positive donors. In contrast to other RD1-encoded antigens, Rv3873 was also found to be recognized by a significant proportion of Mycobacterium bovis BCG-vaccinated donors. Epitope mapping performed with overlapping peptides revealed a broad pattern of T-cell recognition comprising both TB-specific epitopes and epitopes also recognized by BCG-vaccinated donors. The immunodominant epitope (residues 118 to 135) for both TB patients and BCG-vaccinated individuals was found to be highly conserved among a large number of PPE family members.

The NblAI protein from the filamentous cyanobacterium Tolypothrix PCC 7601: regulation of its expression and interactions with phycobilisome components.

Cyanobacteria respond to changes in light or nutrient availability by modifications in their photosynthetic light harvesting antenna. In unicellular cyanobacteria a small polypeptide (NblA) is required for phycobilisome degradation following environmental stresses. In the filamentous strain Tolypothrix sp. PCC 7601 the nblAI gene, encoding a NblA homologue, is located upstream of the operon coding for phycoerythrin (cpeBA). The nblAI transcripts all originate from a single transcription start point; their intracellular levels vary according to nitrogen regimes but not with light spectral quality. Using recombinant His-tagged NblAI protein, we found that in vitro NblAI has affinity for both phycocyanin and phycoerythrin subunits from Tolypothrix sp. PCC 7601, but not for allophycocyanin from this cyanobacterium or for phycobiliproteins from other cyanobacterial species. We also observed that although nblAI is mainly expressed under nitrogen starvation, NblAI polypeptides are always present in the cell; a significant portion of them co-purify with phycobilisome preparations but only if cells were grown under red light. Our data indicate that NblAI attaches to the phycobilisomes even under non-inducing conditions and suggest a preferential affinity of NblAI for phycocyanin.

Juvenile hormone acid methyltransferase: a key regulatory enzyme for insect metamorphosis.

Juvenile hormone (JH) acid methyltransferase (JHAMT) is an enzyme that converts JH acids or inactive precursors of JHs to active JHs at the final step of JH biosynthesis pathway in insects. By fluorescent mRNA differential display, we have cloned a cDNA encoding JHAMT from the corpora allata (CA) of the silkworm, Bombyx mori (BmJHAMT). The BmJHAMT cDNA encodes an ORF of 278 aa with a calculated molecular mass of 32,544 Da. The predicted amino acid sequence contains a conserved S-adenosyl-l-methionine (SAM) binding motif found in the family of SAM-dependent methyltransferases. Purified N-terminal 6xHis-tagged recombinant BmJHAMT protein expressed in Escherichia coli catalyzed conversion of farnesoic acid and JH acids I, II, and III to their cognate methyl esters in the presence of SAM, confirming that this cDNA encodes a functional JHAMT. Putative orthologs, DmJHAMT and AgJHAMT, were identified from the genome sequence of the fruit fly Drosophila melanogaster, and a malaria vector, Anopheles gambiae, respectively. Northern blot and quantitative RT-PCR analyses revealed that the BmJHAMT gene was expressed specifically in the CA throughout the third and fourth instar. At the beginning of the last (fifth) instar, the expression level of BmJHAMT declined rapidly and became undetectable by day 4 and remained so until pupation. Correlation of the BmJHAMT gene expression and the JH biosynthetic activity in the CA suggests that the transcriptional suppression of the BmJHAMT gene is crucial for the termination of JH biosynthesis in the CA, which is a prerequisite for the initiation of metamorphosis.

Mammalian NADH diphosphatases of the Nudix family: cloning and characterization of the human peroxisomal NUDT12 protein

The human NUDT12 Nudix hydrolase has been expressed in insect cells from a baculovirus vector as a His-tagged recombinant protein. In vitro, it efficiently hydrolyses NAD(P)H to NMNH and AMP (2',5'-ADP), and diadenosine diphosphate to AMP. It also has activity towards NAD(P)(+), ADP-ribose and diadenosine triphosphate. K (m) values for NADH, NADPH and NAD(+) are 11, 16 and 190 microM and k (cat) values are 11, 16 and 10.5 s(-1) respectively. Thus, like other NADH diphosphatases of the Nudix family, NUDT12 has a marked substrate preference for the reduced nicotinamide nucleotides. Optimal activity was supported by 50 microM Mn(2+) ions in vitro, with 3-fold lower activity at 0.4 mM Mg(2+). Expression of NUDT12 as a C-terminal fusion to green fluorescent protein revealed that it was targeted to peroxisomes by the C-terminal tripeptide PNL acting as a novel type 1 peroxisomal targeting signal. Deletion of PNL resulted in diffuse cellular fluorescence. In addition, C-terminal, but not N-terminal, fusions with or without the PNL signal accumulated in large, unidentified cytoplasmic structures. NUDT12 may act to regulate the concentration of peroxisomal nicotinamide nucleotide cofactors required for oxidative metabolism in this organelle.

Two-step purification of His-tagged Nef protein in native condition using heparin and immobilized metal ion affinity chromatographies

The Nef protein encoded by human immunodeficiency virus type 1 (HIV-1) has been shown to be an important factor of progression of viral growth and pathogenesis in both in vitro and in vivo. The lack of a simple procedure to purify Nef in its native conformation has limited molecular studies on Nef function. A two-step procedure that includes heparin and immobilized metal ion affinity chromatographies (IMACs) was developed to purify His-tagged Nef (His(6)-Nef) expressed in bacteria in native condition. During the elaboration of this purification procedure, we identified two closely SDS-PAGE-migrating contaminating bacterial proteins, SlyD and GCHI, that co-eluted with His(6)-Nef in IMAC in denaturing condition and developed purification steps to eliminate these contaminants in native condition. Overall, this study describes a protocol that allows rapid purification of His(6)-Nef protein expressed in bacteria in native condition and that removes metal affinity resin-binding bacterial proteins that can contaminate recombinant His-tagged protein preparation.

Evaluation of chromatographic recycling for imidazole used in the chromatographic purification of His-tag recombinant proteins

The aim of this work was to test a recycling method for imidazole used in immobilized metal affinity chromatography (IMAC) as eluent for recombinant histidine-tag (His-tag) protein. After evaluating two supports, the method was optimized with a mixture of bovine serum albumin, sodium chloride and imidazole. Recycling was performed with an eluate fraction from IMAC of His-tag enhanced green fluorescent protein produced in our laboratory and pure imidazole was recovered in water and was analyzed after being freeze-dried. The imidazole was then reused as eluent in IMAC without any modification in its structure or behavior. This procedure can be used for large-scale chromatography.

A novel type of replicative enzyme harbouring ATPase, primase and DNA polymerase activity.

Although DNA replication is a process common in all domains of life, primase and replicative DNA polymerase appear to have evolved independently in the bacterial domain versus the archaeal/eukaryal branch of life. Here, we report on a new type of replication protein that constitutes the first member of the DNA polymerase family E. The protein ORF904, encoded by the plasmid pRN1 from the thermoacidophile archaeon Sulfolobus islandicus, is a highly compact multifunctional enzyme with ATPase, primase and DNA polymerase activity. Recombinant purified ORF904 hydrolyses ATP in a DNA-dependent manner. Deoxynucleotides are preferentially used for the synthesis of primers approximately 8 nucleotides long. The DNA polymerase activity of ORF904 synthesizes replication products of up to several thousand nucleotides in length. The primase and DNA polymerase activity are located in the N-terminal half of the protein, which does not show homology to any known DNA polymerase or primase. ORF904 constitutes a new type of replication enzyme, which could have evolved independently from the eubacterial and archaeal/eukaryal proteins of DNA replication.

Functional Characterization and Localization of Acetyl-CoA Hydrolase, Ach1p, in Saccharomyces cerevisiae

Acetyl-CoA hydrolase (Ach1p), catalyzing the hydrolysis of acetyl-CoA, is presumably involved in regulating intracellular acetyl-CoA or CoASH pools; however, its intracellular functions and distribution remain to be established. Using site-directed mutagenesis analysis, we demonstrated that the enzymatic activity of Ach1p is dependent upon its putative acetyl-CoA binding sites. The ach1 mutant causes a growth defect in acetate but not in other non-fermentable carbon sources, suggesting that Ach1p is not involved in mitochondrial biogenesis. Overexpression of Ach1p, but not constructs containing acetyl-CoA binding site mutations, in ach1-1 complemented the defect of acetate utilization. By subcellular fractionation, most of the Ach1p in yeast was distributed with mitochondria and little Ach1p in the cytoplasm. By immunofluorescence microscopy, we show that Ach1p and acetyl-CoA binding site-mutated constructs, but not its N-terminal deleted construct, are localized in mitochondria. Moreover, the onset of pseudohyphal development in homozygote ach1-1 diploids was abolished. We infer that Ach1p may be involved in a novel acetyl-CoA biogenesis and/or acetate utilization in mitochondria and thereby indirectly affect pseudohyphal development in yeast.

Evaluation of chromatographic recycling for imidazole used in the chromatographic purification of His-tag recombinant proteins

Evaluation of chromatographic recycling for imidazole used in the chromatographic purification of His-tag recombinant proteins

New N-RAP-binding partners alpha-actinin, filamin and Krp1 detected by yeast two-hybrid screening: implications for myofibril assembly.

N-RAP, a muscle-specific protein concentrated at myotendinous junctions in skeletal muscle and intercalated disks in cardiac muscle, has been implicated in myofibril assembly. To discover more about the role of N-RAP in myofibril assembly, we used the yeast two-hybrid system to screen a mouse skeletal muscle cDNA library for proteins capable of binding N-RAP in a eukaryotic cell. From yeast two-hybrid experiments we were able to identify three new N-RAP binding partners: alpha-actinin, filamin-2, and Krp1 (also called sarcosin). In vitro binding assays were used to verify these interactions and to identify the N-RAP domains involved. Three regions of N-RAP were expressed as His-tagged recombinant proteins, including the nebulin-like super repeat region (N-RAP-SR), the N-terminal LIM domain (N-RAP-LIM), and the region of N-RAP in between the super repeat region and the LIM domain (N-RAP-IB). We detected significant alpha-actinin binding to N-RAP-IB and N-RAP-LIM, filamin binding to N-RAP-SR, and Krp1 binding to N-RAP-SR and N-RAP-IB. During myofibril assembly in cultured chick cardiomyocytes, N-RAP and filamin appear to co-localize with alpha-actinin in the earliest myofibril precursors found near the cell periphery, as well as in the nascent myofibrils that form as these structures fuse laterally. In contrast, Krp1 is not localized until late in the assembly process, when it appears at the periphery of myofibrils that appear to be fusing laterally. The results suggest that sequential recruitment of N-RAP binding partners may serve an important role during myofibril assembly.

Expression in Escherichia coli of human ARHGAP6 gene and purification of His-tagged recombinant protein.

In this report we describe cloning and expression of human Rho GTPase activating protein (ARHGAP6) isoform 4 in Escherichia coli cells as a fusion protein with 6xHis. We cloned the ARHGAP6 cDNA into the bacterial expression vector pPROEX-1. Induction of the 6xHis-ARHGAP6 protein in BL21(DE3) and DH5alpha cells caused lysis of the cells irrespective of the kind of culture medium used. Successful expression of the fusion protein was obtained in the MC4100Deltaibp mutant strain lacking the small heat-shock proteins IbpA and IbpB. Reasonable yield was obtained when the cells were cultured in Terrific Broth + 1% glucose medium at 22 degrees C for 16 h. The optimal cell density for expression of soluble 6xHis-ARHGAP6 protein was at A(600) about 0.5. Under these conditions over 90% of the fusion protein was present in a soluble form. The 6xHis-ARHGAP6 protein was purified to near homogeneity by a two step procedure comprising chromatography on Ni-nitrilotriacetate and cation exchange columns. The expression system and purification procedure employed made it possible to obtain 1-2 mg of pure 6xHis-ARHGAP6 protein from 300 ml (1.5 g of cells) of E. coli culture.

Expression in Escherichia coli of human ARHGAP6 gene and purification of His-tagged recombinant protein.

In this report we describe cloning and expression of human Rho GTPase activating protein (ARHGAP6) isoform 4 in Escherichia coli cells as a fusion protein with 6xHis. We cloned the ARHGAP6 cDNA into the bacterial expression vector pPROEX-1. Induction of the 6xHis-ARHGAP6 protein in BL21(DE3) and DH5alpha cells caused lysis of the cells irrespective of the kind of culture medium used. Successful expression of the fusion protein was obtained in the MC4100Deltaibp mutant strain lacking the small heat-shock proteins IbpA and IbpB. Reasonable yield was obtained when the cells were cultured in Terrific Broth + 1% glucose medium at 22 degrees C for 16 h. The optimal cell density for expression of soluble 6xHis-ARHGAP6 protein was at A(600) about 0.5. Under these conditions over 90% of the fusion protein was present in a soluble form. The 6xHis-ARHGAP6 protein was purified to near homogeneity by a two step procedure comprising chromatography on Ni-nitrilotriacetate and cation exchange columns. The expression system and purification procedure employed made it possible to obtain 1-2 mg of pure 6xHis-ARHGAP6 protein from 300 ml (1.5 g of cells) of E. coli culture.

Identification of a mitochondrial nucleoside diphosphate kinase from the green alga Dunaliella tertiolecta.

We isolated a full-length cDNA encoding a nucleoside diphosphate (NDP) kinase from a Dunaliella tertiolecta cDNA library by homology cloning and rapid amplification of cDNA ends-PCR. The cDNA sequence, consisting of 840 bp, contained an open reading frame coding for a 221-amino acid protein. The predicted 24-kDa protein was named DtNDK1. It possesses all the residues involved in nucleotide binding and catalysis and, in its long N-terminus, contains putative mitochondrial targeting peptides. The full-length pre-protein expressed in Escherichia coli as a recombinant N-terminally His-tagged protein was retained in inclusion bodies, totally devoid of NDP kinase activity. Upon expression in yeast cells, the full-length protein His-tagged at the C-terminus was found processed in a soluble form that was lacking the first 67 amino acids from the N-terminus. The mature protein, which was purified by affinity chromatography to near homogeneity, showed NDP kinase activity. Confocal microscopy on yeast cells expressing the recombinant protein revealed the specific mitochondrial localization of DtNDK1 labeled at the C-terminus with green fluorescent protein.

Localization and function of a calmodulin-apocalmodulin-binding domain in the N-terminal part of the type 1 inositol 1,4,5-trisphosphate receptor.

Calmodulin (CaM) is a ubiquitous protein that plays a critical role in regulating cellular functions by altering the activity of a large number of proteins, including the d-myo-inositol 1,4,5-trisphosphate (IP3) receptor (IP3R). CaM inhibits IP3 binding in both the presence and absence of Ca2+ and IP3-induced Ca2+ release in the presence of Ca2+. We have now mapped and characterized a Ca2+-independent CaM-binding site in the N-terminal part of the type 1 IP3R (IP3R1). This site could be responsible for the inhibitory effects of CaM on IP3 binding. We therefore expressed the N-terminal 581 amino acids of IP3R1 as a His-tagged recombinant protein, containing the functional IP3-binding pocket. We showed that CaM, both in the presence and absence of Ca2+, inhibited IP3 binding to this recombinant protein with an IC50 of approx. 2 microM. Deletion of the N-terminal 225 amino acids completely abolished the effects of both Ca2+ and CaM on IP3 binding. We mapped the Ca2+-independent CaM-binding site to a recombinant glutathione S-transferase fusion protein containing the first 159 amino acids of IP3R1 and then made different synthetic peptides overlapping this region. We demonstrated that two synthetic peptides matching amino acids 49-81 and 106-128 bound CaM independently of Ca2+ and could reverse the inhibition of IP3 binding caused by CaM. This suggests that these sequences are components of a discontinuous Ca2+-independent CaM-binding domain, which is probably involved in the inhibition of IP3 binding by CaM.

Integrin Cytoplasmic Domain-associated Protein 1α (ICAP-1α) Interacts Directly with the Metastasis Suppressor nm23-H2, and Both Proteins Are Targeted to Newly Formed Cell Adhesion Sites upon Integrin Engagement

Cell adhesion-dependent signaling implicates cytoplasmic proteins interacting with the intracellular tails of integrins. Among those, the integrin cytoplasmic domain-associated protein 1alpha (ICAP-1alpha) has been shown to interact specifically with the beta(1) integrin cytoplasmic domain. Although it is likely that this protein plays an important role in controlling cell adhesion and migration, little is known about its actual function. To search for potential ICAP-1alpha-binding proteins, we used a yeast two-hybrid screen and identified the human metastatic suppressor protein nm23-H2 as a new partner of ICAP-1alpha. This direct interaction was confirmed in vitro, using purified recombinant ICAP-1alpha and nm23-H2, and by co-immunoprecipitation from CHO cell lysates over-expressing ICAP-1alpha. The physiological relevance of this interaction is provided by confocal fluorescence microscopy, which shows that ICAP-1alpha and nm23-H2 are co-localized in lamellipodia during the early stages of cell spreading. These adhesion sites are enriched in occupied beta(1) integrins and precede the formation of focal adhesions devoid of ICAP-1alpha and nm23-H2, indicating the dynamic segregation of components of matrix adhesions. This peripheral staining of ICAP-1alpha and nm23-H2 is only observed in cells spreading on fibronectin and collagen and is absent in cells spreading on poly-l-lysine, vitronectin, or laminin. This is consistent with the fact that targeting of both ICAP-1alpha and nm23-H2 to the cell periphery is dependent on beta(1) integrin engagement rather than being a consequence of cell adhesion. This finding represents the first evidence that the tumor suppressor nm23-H2 could act on beta(1) integrin-mediated cell adhesion by interacting with one of the integrin partners, ICAP-1alpha.

CcaR is an autoregulatory protein that binds to the ccaR and cefD-cmcI promoters of the cephamycin C-clavulanic acid cluster in Streptomyces clavuligerus.

The putative regulatory CcaR protein, which is encoded in the beta-lactam supercluster of Streptomyces clavuligerus, has been partially purified by ammonium sulfate precipitation and heparin affinity chromatography. In addition, it was expressed in Escherichia coli, purified as a His-tagged recombinant protein (rCcaR), and used to raise anti-rCcaR antibodies. The partially purified CcaR protein from S. clavuligerus was able to bind DNA fragments containing the promoter regions of the ccaR gene itself and the bidirectional cefD-cmcI promoter region. In contrast, CcaR did not bind to DNA fragments with the promoter regions of other genes of the cephamycin-clavulanic acid supercluster including lat, blp, claR, car-cyp, and the unlinked argR gene. The DNA shifts obtained with CcaR were prevented by anti-rCcaR immunoglobulin G (IgG) antibodies but not by anti-rabbit IgG antibodies. ccaR and the bidirectional cefD-cmcI promoter region were fused to the xylE reporter gene and expressed in Streptomyces lividans and S. clavuligerus. These constructs produced low catechol dioxygenase activity in the absence of CcaR; activity was increased 1.7- to 4.6-fold in cultures expressing CcaR. Amplification of the ccaR promoter region lacking its coding sequence in a high-copy-number plasmid in S. clavuligerus ATCC 27064 resulted in a reduced production of cephamycin C and clavulanic acid, by 12 to 20% and 40 to 60%, respectively, due to titration of the CcaR regulator. These findings confirm that CcaR is a positively acting autoregulatory protein able to bind to its own promoter as well as to the cefD-cmcI bidirectional promoter region.

Recombinant nucleoprotein-based enzyme-linked immunosorbent assay for detection of immunoglobulin G antibodies to Crimean-Congo hemorrhagic fever virus.

The full-length nucleoprotein of Crimean-Congo hemorrhagic fever virus (CCHFV; 482 amino acid residues) was expressed as a His-tagged recombinant protein (His-CCHFV rNP) in the baculovirus system. The His-CCHFV rNP was efficiently expressed in insect cells and purified by Ni(2+) column chromatography. Using this substrate, an immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) was developed. We evaluated the sensitivity and specificity of the IgG ELISA, using serum samples previously determined to be antibody positive or negative by immunofluorescence tests on CCHFV-infected Vero E6 cells. We found very good correlation between the two tests: 87% for the positive sera (13 of 15) and 99% for the negative sera (107 of 108). These results indicate that the new IgG ELISA using His-CCHFV rNP has high sensitivity and specificity for detecting CCHFV antibodies. The CCHF patients' sera with high titers reacted only with the NP fragment containing amino acid residues between 201 and 306 in Western blotting. It is known that amino acid homologies are high in this region among various isolates. Thus, it is expected that this ELISA can detect antibodies not only for Chinese strains of CCHFV but also for other strains circulating in the world. These results suggest that the IgG ELISA system developed with the recombinant CCHFV NP is a valuable tool for diagnosis and epidemiological investigations of CCHFV infections.

The production of human papillomavirus type 16 L1 vaccine product from Escherichia coli inclusion bodies.

The major capsid protein L1 of the human papillomavirus type 16 (HPV16) has been previously expressed recombinantly in Escherichia coli cells as inclusion bodies (IBs). The HPV16 L1 protein offers potential as a vaccine candidate against cervical cancer, but the reported E. coli process is limited in its ability to economically produce significant quantities of material. In this study, a scaleable laboratory process for the purification of recombinant His-tagged L1 protein and its processing to give an immunogenic product is developed. The performances of ion-exchange chromatography (IEX) and immobilised metal affinity chromatography (IMAC) for the purification of L1 protein in the presence of concentrated denaturant are compared. IEX was found to be superior to IMAC when taking into account the complexity of operation, cost of adsorbent, selectivity and purity of the final product. Following purification, reduction of denaturant concentration was performed by dilution to yield a product suitable for formulation. The simplicity and ease of scale-up of dilution makes it an attractive option for process scale production and superior to the existing approach employing dialysis. It was found that direct dilution of denaturant into suitable buffer can give rise to products which have neutralising conformational epitopes identified by strong antibody-binding properties, as assessed by ELISA with a conformational monoclonal antibody. Analysis of the results showed negative main effects of protein concentration and PEG addition on antibody-binding yields, but positive main effects of the addition of detergent and L-arginine to the buffer. The diluted product had antigenic properties as assessed by ELISA and may be formulated easily for use by diafiltration and the addition of adjuvant. This work demonstrates the feasibility of producing viral vaccines using E. coli and scaleable unit operations.