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Results for “Recombinant expression”

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PubMed1997

Microheterogeneity of the oligosaccharides carried by the recombinant bovine lactoferrin expressed in Mamestra brassicae cells.

The development of therapeutic glycoprotein production using the baculovirus expression system depends on the ability of insect cell lines to reproduce site specific mammalian-like N-glycans. A combination of 1H-NMR and mass spectrometry techniques (MALD-MS, ES-MS, and CID-MS-MS) allowed us to elucidate the N-linked oligosaccharides microheterogeneity on three different N-glycosylation sites, Asn233, Asn476, and Asn545, of a baculovirus-expressed recombinant bovine lactoferrin produced in Mamestra brassicae. Two families of N-glycan structures have been found: first, oligomannosidic glycans (Man[9-5]GlcNAc2) and secondly, short truncated partially fucosylated glycans (Man(3-2)[Fuc(0-1)]GlcNAc2). These results indicate that Mamestra brassicae cell line is not able to synthesize complex N-glycans, even if an alpha1,6-linked fucose residue is frequently present on the asparagine-bound N-acetylglucosamine residue of short truncated structures. Nevertheless, we have shown that Mamestra brassicae ensures the same N-glycosylation pattern as found on natural bovine lactoferrin showing the same distribution between complex and high-mannose type glycans on the different glycosylation sites. Sites which are naturally occupied by high-mannose glycans (Asn233 and Asn545) are substituted essentially by the same type of N-glycans in the recombinant counterpart, and the site Asn476,which carries sialylated complex type chains in the natural glycoprotein, is substituted by short, truncated, partially fucosylated chains in Mamestra brassicae-expressed bovine lactoferrin. These various results lead us to the conclusion that bovine lactoferrin is an interesting model to determine the potential of glycosylation of the baculovirus/insect cell expression systems.

Amino Acid Sequence↗

PubMed2003

[Compatible-solute-supported periplasmic expression of recombinant immunotoxins and the effects of glucose thereby].

Bacteria protect themselves against high external osmolality by uptake or synthesis of a limited number of so-called compatible solutes. Under such extreme conditions, combine factors can facilitate the foreign recombinant protein expression. In our experiment, we added additional NaCl and two compatible solutes (glycine betaine and sorbitol) into the medium. The recombinant immunotoxin BDI(scFv)-PE38 was expressed mainly in soluble form and can be purified by Ni2+-NTA agarose. We also found the time when the glucose was added into the medium greatly affected the expression level of the recombinant immunotoxin. This suggested that glucose played an important role in the expression system.

English Abstract↗

PubMed2004

[Bioassay of the soluble human tumor necrosis factor receptor I recombinant plasmid expression in vitro].

OBJECTIVE: To detect the expression of recombinant plasmid PcDNA3.1-sTNFRI in vitro and evaluate the bioactivity of expressed sTNFRI. METHODS: CHO cells were transfected with recombinant plasmid PcDNA3.1-sTNFRI by liposome. sTNFRI in cell culture supernatant was detected by ELISA and sTNFRI blockage of TNF-alpha cytotoxicity in L929 cells evaluated by MTT assay. RESULTS: The expression of sTNFRI in transfected cell culture supernatant was higher than control groups (P < 0.001). The expressed sTNFRI could significantly neutralize TNF-alpha cytotoxicity in L929 cells. CONCLUSIONS: These results showed that the recombinant plasmid PcDNA3.1-sTNFRI can be expressed in mammalian cells and the recombinant sTNFRI has biological function.

Animals↗

PubMed2005

An efficient strategy for high-throughput expression screening of recombinant integral membrane proteins.

The recombinant expression of integral membrane proteins is considered a major challenge, and together with the crystallization step, the major hurdle toward routine structure determination of membrane proteins. Basic methodologies for high-throughput (HTP) expression optimization of soluble proteins have recently emerged, providing statistically significant success rates for producing such proteins. Experimental procedures for handling integral membrane proteins are generally more challenging, and there have been no previous comprehensive reports of HTP technology for membrane protein production. Here, we present a generic and integrated parallel HTP strategy for cloning and expression screening of membrane proteins in their detergent solubilized form. Based on this strategy, we provide overall success rates for membrane protein production in Escherichia coli, as well as initial benchmarking statistics of parameters such as expression vectors, strains, and solubilizing detergents. The technologies were applied to 49 E. coli integral membrane proteins with human homologs and revealed that 71% of these proteins could be produced at sufficient levels to allow milligram amounts of protein to be relatively easily purified, which is a significantly higher success rate than anticipated. We attribute the high success rate to the quality and robustness of the methodology used, and to introducing multiple parameters such as different vectors, strains, and detergents. The presented strategy demonstrates the usefulness of HTP technologies for membrane protein production, and the feasibility of large-scale programs for elucidation of structure and function of bacterial integral membrane proteins.

Chromatography, Gel↗

PubMed2006

Recombinant human antithrombin expressed in Chinese hamster ovary cells shows in vivo efficacy on rat DIC model similarly to plasma-derived antithrombin regardless of different N-glycosylation.

Plasma-derived human antithrombin (pAT) is used for the treatments of disseminated intravascular coagulation (DIC) and hereditary antithrombin deficiencies. We expressed recombinant human antithrombin (rAT) in Chinese hamster ovary (CHO) cells. The purified rAT is composed of 55% alpha-isoform and 45% beta-isoform. The structure of the N-linked oligosaccharides of rAT is the same biantennary complex type as previously found in pAT with less sialylated on the non-reducing ends. Most of the oligosaccharides of rAT are fucosylated at the reducing ends of N-acetylglucosamine, while those of pAT are not fucosylated. Despite of the difference in sialylation and fucosylation of the oligosaccharide units, rAT and pAT showed indistinguishable heparin cofactor and progressive activities, and they bound to thrombin in a one-to-one stoichiometric manner. In lipopolysaccharide (LPS)-induced and thromboplastin-induced DIC rat models, rAT reduced fibrinogen and platelet consumption to a similar extent with pAT. In LPS-induced DIC model, both ATs similarly restrained the increase of alanine aminotransferase and aspartate aminotransferase activities. Finally, pharmacokinetic analysis showed that both ATs had similar half-lives in the circulation of normal rats. Together, the present study demonstrated that rAT prepared in CHO cells has potential for a substitute of pAT in therapeutic use.

Alanine Transaminase↗

PubMed2002

Preparation of active recombinant cathepsin K expressed in bacteria as inclusion body.

Human cathepsin K (EC 3.4.22.38) is a member of the cysteine protease family with high primary sequence homology to cathepsins S, L, and B. It has been shown that cathepsin K plays a major role in the resorption of the bone matrix by osteoclasts. Cathepsin K has a potential as a drug target for the diseases related to bone matrix metabolism such as osteoporosis. We have expressed recombinant human procathepsin K in Escherichia coli as inclusion bodies. Purified procathepsin K had size of 38kDa which is in agreement with the predicted mass of the construct. Refolding was done by rapid dilution into 50mM Tris-HCl, pH 8.0 buffer containing 5mM EDTA, 10 mM GSH, 1mM GSSG, 0.7 M L-arginine, 0.5 M NaCl, and 1% CHAPS and further dialysis against 25 mM Tris-HCl, pH 8.0 containing 0.5 M NaCl. Mature active cathepsin K was prepared from refolded procathepsin K by incubating at 40 degrees C in pH 4.0 buffers with or without pepsin or cysteine. The presence of pepsin or cysteine in autocatalysis buffer did not have effect on the degree of conversion of nascent to mature cathepsin K, but reduced the autocatalysis time slightly. Proteolytic activity was confirmed using synthetic substrate, and Western blotting identified mature cathepsin K. Active cathepsin K had type I and II collagenolytic activities which could be inhibited by E-64.

Catalysis↗

PubMed2003

A novel method for increasing the expression level of recombinant proteins.

Expression of recombinant proteins is an important step towards elucidating the functions of many genes discovered through genomic sequencing projects. It is also critical for validating gene targets and for developing effective therapies for many diseases. Here we describe a novel method to express recombinant proteins that are extremely difficult to produce otherwise. The increased protein expression level is achieved by using a fusion partner, MTB32-C, which is the carboxyl terminal fragment of the Mycobacterium tuberculosis antigen, MTB32 (Rv0125). By fusing MTB32-C to the N-termini of target genes, we have demonstrated significant enhancement of recombinant protein expression level in Escherichia coli. The inclusion of a 6xHis tag and the 128-amino acid of MTB32-C will add 13.5 kDa to the fusion molecule. Comparison of the mRNA levels of the fusion and non-fusion proteins indicated that the increased fusion protein expression may be regulated at translational or post-translational steps. There are many potential applications for the generated fusion proteins. For example, MTB32-C fusion proteins have been used successfully as immunogens to generate both polyclonal and monoclonal antibodies. These antibodies have been used to characterize cellular localization of the proteins and to validate gene targets at protein level. In addition, these antibodies may be useful in diagnostic and therapeutic applications for many diseases. If desired, the MTB32-C portion in the fusion protein can be removed after protein expression, making it possible to study protein structure and function as well as to screen for potential drugs. Thus, this novel fusion expression system has become a powerful tool for many applications.

Amino Acid Sequence↗

PubMed1995

High-level expression of recombinant porcine LH receptor in baculovirus-infected insect cells or caterpillars.

Porcine LH receptor ectodomain was overexpressed in insect cells and lepidopteran larvae using the recombinant baculovirus expression system. A low multiplicity of infection yielded the largest active production, of approximately 10(7) receptors/cell or 3 micrograms active receptor/mg total protein in infected cells. The truncated ectodomain solubilized with Triton X-100 bound its ligand with a high affinity which was comparable with that of the native membrane receptor. Increasing the multiplicity of infection resulted in an optimum protein production of 0.6 mg receptor/mg total protein in infected cells. This receptor was largely inactive, probably trapped within aggregation pools. Active receptor could be recovered by dilution of the samples. No secretion of recombinant receptor was ever observed whatever the conditions of infection. Expression of the recombinant receptor in insect larvae was also tested. This low-cost system failed both to increase the amount of active receptor and to induce secretion into the haemolymph. Two methods remain for producing sizeable amounts of active receptor with this baculovirus/insect cell system. One relies on immunoaffinity purification of the active protein and requires large-scale production, and the other is based on the purification of overexpressed inactive receptor followed by renaturation.

Amino Acid Sequence↗

PubMed2006

A comparative study of different vector designs for the mammalian expression of recombinant IgG antibodies.

Monoclonal antibodies (Mab) are the fastest growing group of biopharmaceuticals in development. For production in mammalian cells, the four polypeptide chains of the immunoglobulin diheterotetramer must be assembled prior to exit from the endoplasmic reticulum. Various recombinant Mab expression vectors have been developed utilizing mono-and bicistronic expression cassettes encoded on one or two plasmids. However, there are only few studies providing information on the type of vector design optimal for stable or transient production of recombinant IgG. Consequently, in this study, we have constructed a series of mammalian expression vectors for the production of recombinant human or chimeric IgG antibodies with different expression cassette designs. Versions for monocistronic and bicistronic expression with different promoters and cistron arrangements were generated. Antibody production levels were evaluated in transiently transfected 293T and CHO-K1 cells. Furthermore, stable CHO cell lines were generated and analyzed for antibody production levels and stability. Our results indicate that compared to monocistronic expression, EMCV IRES-mediated bicistronic expression constructs yield similar antibody expression levels and show long-term stability in CHO cell lines. Addition of a third cistron encoding YFP was shown to facilitate screening and isolation of clones using a FACS sorter.

5' Untranslated Regions↗

PubMed2001

Mechanisms of loss of foreign gene expression in recombinant vesicular stomatitis viruses.

We investigated the stability and mechanisms of loss of foreign gene expression in two recombinant vesicular stomatitis viruses (VSVs). A recombinant expressing the cellular CD4 protein exhibited remarkable stability of foreign gene expression. However, after 26 sequential passages, a mutant no longer expressing CD4 was recovered from the virus stock. Sequencing of the CD4 coding region in this mutant revealed a single nucleotide deletion causing a frameshift and termination of protein synthesis. A second VSV recombinant expressing the measles virus F protein grew poorly and exhibited extreme instability of expression of the F protein. Expression of F protein was lost rapidly through mutations of the upstream transcription termination site from (3')AUAC(5') to (3')AUAU(5'), as well as lengthening of the subsequent U(7) tract that is the template for poly(A) addition to VSV G mRNA. Such mutations resulted in fusion of the F mRNA to the 3' end of the G mRNA, making the F protein translation initiation codon inaccessible. We suggest that the VSV polymerase is error prone during replication of the U(7) tract, providing a rapid means for complete elimination of expression of proteins that are toxic to the virus life cycle.

Animals↗

PubMed2007

[Construction and prokaryotic expression of recombinant gene EGFRvIII HBcAg and immunogenicity analysis of the fusion protein].

AIM: To construct recombinant prokaryotic expression plasmid pET28a(+)/c-PEP-3-c and evaluate the immunogenicity of the fusion protein. METHODS: cDNA fragment encoding PEP-3 was obtained from pGEM-T Easy/PEP-3 and inserted into recombinant plasmid pGEMEX/HBcAg. Then it was subcloned in prokaryotic expression vector and transformed into E.coli BL21(DE3). The fusion protein was expressed by inducing IPTG and purified by Ni(2+)-NTA affinity chromatography. BALB/c mice were immunized with fusion protein and the antibody titre was determined by indirect ELISA. RESULTS: The recombinant gene was confirmed to be correct by restriction enzyme digestion and DNA sequencing. After prokaryotic expression, fusion protein existed in sediment and accounted for 56% of all bacterial lysate. The purified product accounted for 92% of all protein and its concentration was 8 g/L. The antibody titre in blood serum reached 1:16 000 after the fourth immunization and reached 1:2.56x10(5) after the sixth immunization. The titre of anti-PEP-3 antibody reached 1:1.28x10(5) and the titre of anti-HBcAg antibody was less than 1:4x10(3). CONCLUSION: Fusion gene PEP-3-HBcAg is highly expressed in E.coli BL21. The expressed fusion protein can induce neutralizing antibody with high titer and specificity, which lays a foundation for the study of genetically engineering vaccine for malignant tumors with the high expression of EGFRvIII.

Animals↗

PubMed1996

Optimization of recombinant gene expression in Escherichia coli.

The major targets for improvement of recombinant expression efficiency in Escherichia coli are gene dosage, transcription and, to some extent, translation. In order to evaluate the relative importance of these factors, the kinetics of specific mRNA compared to product formation was studied for different widely used expression systems, producing recombinant human superoxide dismutase. For a system employing phage T7 RNA polymerase, where a high level of recombinant protein expression puts a high metabolic burden on the cells, it was shown that transcription is not the limiting factor. To improve the translation rate of a common vector based on the tac promoter, the Shine-Dalgarno (SD) sequence was mutated towards stronger homology to the anti-SD sequence of the E. coli 16S rRNA. A 12.2-fold increase in protein yield was accompanied by a 4.3-fold increase in specific mRNA, indicating that transcription of the recombinant gene is coupled to translation. As this coupling amplifies the detrimental effect of a low-efficiency ribosomal binding site, much attention should be paid to translation initiation when optimizing a recombinant protein production system. Finally, reasons for the high expression level before induction are discussed, and first results towards reducing it are presented.

Base Sequence↗

PubMed1993

Immunogenicity in mice of tandem repeats of an epitope from herpes simplex gD protein when expressed by recombinant adenovirus vectors.

The antigenic and immunogenic potential was examined of human adenovirus type 5 (Ad) recombinants carrying and expressing from one to four tandem repeats of a linear neutralizing epitope from the gD protein of herpes simplex virus type 1 (HSV-1) as a fusion with the beta-galactosidase protein. The fusion proteins produced by these Ad vectors in infected cell culture reacted with a herpes simplex virus (HSV) epitope-specific monoclonal antibody to a degree dependent on the number of epitope repeats in the protein. Mice immunized by intraperitoneal injection of the Ad vectors developed an anti-HSV immune response as measured by ELISA and by HSV-1 neutralization assays. The mean antibody titre induced by a single injection of the Ad vector increased with the number of epitope repeats expressed by the recombinant. Any animal that had developed a serum-neutralizing titre of at least 1:80 survived challenge with a normally lethal dose of HSV-2 administered by the intraperitoneal route. Recombinant vectors expressing four repeats of the HSV epitope were as effective in antibody induction and protection as an adenovirus vector carrying and expressing the entire HSV gD protein. These results suggest that the expression of tandem repeats of appropriate epitopic sequences by adenovirus vectors may provide a safe and effective method of immunizing against HSV infection.

Adenoviruses, Human↗

PubMed1995

Induction of antibodies protecting against transmissible gastroenteritis coronavirus (TGEV) by recombinant adenovirus expressing TGEV spike protein.

Ten recombinant adenoviruses expressing either fragments of 1135, 1587, or 3329 nt or the full-length spike gene of transmissible gastroenteritis coronavirus (TGEV) have been constructed. These recombinants produce S polypeptides with apparent molecular masses of 68, 86, 135, and 200 kDa, respectively. Expression of the recombinant antigen driven by Ad5 promoters was inhibited by the insertion of an exogenous SV-40 promoter. Most of the recombinant antigens remain intracytoplasmic in infected cells. All the recombinant-directed expression products contain functional antigenic sites C and B (Gebauer et al., 1991, Virology 183, 225-238). The recombinant antigen of 135 kDa and that of 200 kDa, which represents the whole spike protein, also contain antigenic sites D and A, which have previously been shown to be the major inducers of TGEV-neutralizing antibodies. Interestingly, here we show that recombinant S protein fragments expressing only sites C and B also induced TGEV-neutralizing antibodies. The chimeric Ad5-TGEV recombinants elicited lactogenic immunity in hamsters, including the production of TGEV-neutralizing antibodies. The antisera induced in swine by the Ad5 recombinants expressing the amino-terminal 26% of the spike protein (containing sites C and B) or the full-length spike protein, when mixed with a lethal dose of virus prior to administration to susceptible piglets, delayed or completely prevented the induction of symptoms of disease, respectively.

Adenoviruses, Human↗

PubMed2006

Expression of recombinant proteins lacking methionine as N-terminal amino acid in plastids: human serum albumin as a case study.

Removal of the N-terminal methionine of a protein could be critical for its function and stability. Post-translational modifications of recombinant proteins expressed in heterologous systems may change amino-terminal regions. We studied the expression of mature proteins lacking methionine as the N-terminal amino acid in tobacco chloroplasts, using human serum albumin (HSA) as an example. Two approaches were explored. First, we fused the Rubisco small subunit transit peptide to HSA. This chimeric protein was correctly processed in the stroma of the chloroplast and rendered the mature HSA. The second approach took advantage of the endogenous N-terminal methionine cleavage by methionine aminopeptidase. Study of this protein processing reveals a systematic cleavage rule depending on the size of the second amino acid. Analysis of several foreign proteins expressed in tobacco chloroplasts showed a cleavage pattern in accordance to that of endogenous proteins. This knowledge should be taken into account when recombinant proteins with N-terminus relevant for its function are expressed in plastids.

Amino Acid Sequence↗

PubMed2005

[Construction and prokaryotic expression of recombinant gene c-Abeta-c and the immunogenicity analysis of the fusion protein].

AIM: To construct the recombinant prokaryotic expression plasmid pHGhis/c-Abeta-c and evaluate the immunogenicity of the fusion protein expressed in E.coli DH5 alpha. METHODS: The gene fragments of HBc1-71, HBc88-144 and Abeta(1-42) were amplified by PCR. Then the Abeta(1-42) gene was inserted between HBc1-71 and HBc88-144, yielding the recombinant gene c-Abeta-c. c-Abeta-c gene was cloned into pGEMEX and then subcloned into pHGhis plasmids. c-Abeta-c fusion protein expression in transformed E.coli DH5alpha was induced at 42degrees Celsius. The expressed fusion protein was analyzed by SDS-PAGE. Six BALB/c mice recieved intraperitoneal injection (i.p.) of c-Abeta-c fusion protein purified by saturated ammonium sulfate. The anti-Abeta antibody was detected by indirect ELISA. RESULTS: The recombinant gene was confirmed by restriction enzyme digestion and DNA sequencing. After temperature induction, fusion protein was expressed and existed in the sediment of the bacterial lysate. The expression level was 16% of all the proteins in the sediment. After 3 times of immunization, the titer of anti-Abeta antibody in sera of BALB/c mice reached up to 1:16,000, while the anti-HBc antibody was undetectable. CONCLUSION: Recombinant c-Abeta-c gene can be expressed in E.coli DH5 alpha. The expressed protein exists in the sediment of the bacterial lysate and has a strong immunogenicity. This study lays the foundation for the experimental animal study of Alzheimer's disease genetic engineering vaccine.

Amyloid beta-Peptides↗

PubMedDate not supplied

EASE vectors for rapid stable expression of recombinant antibodies.

Over the past 10 years, monoclonal antibodies and antibody fragments have become an increasingly important source of therapeutic molecules in the biotechnology industry. Drug development strategies rely on screening large numbers of candidate molecules in search of an optimized drug candidate. This strategy requires efficient production of ten to a few hundred milligrams of candidate molecules for screening in bioassays and animal models. Typically, this amount of recombinant protein expression involves large numbers of transient transfections or cloning of a recombinant cell line. Both of these approaches are time-consuming and labor-intensive. In this report, we describe the application of an EASE vector system that is capable of generating stable pools of transfected Chinese hamster ovary cells. These pooled populations of cells produce high quantities of antibody candidates without labor-intensive cloning in a 3-5 week time frame. When an optimal drug candidate has been selected, pools generated with EASE-containing vectors can also be used in subsequent cloning steps to make cell lines with improved expression levels. We demonstrate that EASE increases expression in nonamplified pools in addition to increasing amplification and viability of clonal cell lines generated with the EASE-containing vectors compared with pools and cell lines generated without EASE.

Animals↗

PubMed1997

High levels of recombinant CYP3A4 expression in Chinese hamster ovary cells are modulated by coexpressed human P450 reductase and hemin supplementation.

Expression of recombinant cytochrome P450s (P450s) in mammalian cells has been used as a powerful tool to study these enzymes. However, the activity of CYP3A4 expressed in several stable mammalian cell lines was much lower than native enzyme in human liver. The low level of recombinant CYP3A4 may have been due to the low copy number of the cDNA. In addition, the low activity is caused by the low level of P450 reductase in these cells. To achieve high levels of CYP3A4 expression, we employed gene amplification of the CYP3A4 cDNA in Chinese hamster ovary (CHO) cells followed by transfection of the P450 reductase cDNA. Using this strategy, we have obtained a cell line, designated D3A4, with high levels of recombinant CYP3A4. The content of spectrally active P450 was 14 pmol/mg total cellular protein. Hemin treatment increased the P450 content 2-fold. Upon coexpression of P450 reductase in DHR/3A4 cells, enzyme activity of CYP3A4 was stimulated 15-fold, despite a 40% decrease in spectrally active P450. Interestingly, the latter effect was not due to a decrease in CYP3A4 mRNA. Treatment of these cells with hemin, however, counteracted the P450 reductase-mediated decrease of spectrally active P450. These data demonstrate that P450 reductase has a strong influence on the levels of recombinant P450 holoenzyme, possibly by modulating the level of heme in CHO cells. Concomitantly our results show that the gene amplification strategy provides a powerful approach to obtain a high level of functional recombinant P450.

Animals↗