Expression of bovine leukemia virus <i>ENV</i> glycoprotein in insect cells by recombinant baculovirus

Russo, Silvia; Montermini, Laura; Berkovitz-Siman-Tov, Revital; Ponti, W.; Poli, G.

doi:10.1016/s0014-5793(98)00951-x

Cited by 10 publications

(11 citation statements)

References 29 publications

(33 reference statements)

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“…This hypothesis is supported by previous studies: (i) the expression of Env BLV glycoproteins without a signal sequence did not produce a secreted gp51 protein (11,19), and (ii) expression systems in which Env BLV glycoproteins or gp51 are expressed with the signal sequence produced a secreted protein (14,24). Secreted gp51 offers several advantages over intracellular gp51 or gp51 produced by FLK-BLV mammalian cells.…”

Section: Discussionsupporting

confidence: 70%

“…2). All the proteins that migrated faster than native gp51 could represent incomplete and/or incorrectly glycosylated molecules, as is frequently observed in insect cells (16,19) and different mammalian cell lines (1,26).…”

Section: Discussionmentioning

confidence: 89%

“…In recent decades different investigators have described the expression of the BLV Env glycoproteins in heterologous expression systems such as Escherichia coli (2,21,22), Saccharomyces cerevisiae (11), and recombinant vaccinia virus (10,18) systems and, recently, a baculovirus system (9,19). However, the use of recombinant gp51 (rgp51) in an ELISA method has not yet been described.…”

mentioning

confidence: 99%

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Expression of the Bovine Leukemia Virus Envelope Glycoprotein (gp51) by Recombinant Baculovirus and Its Use in an Enzyme-Linked Immunosorbent Assay

Giuseppe

Feliziani

Rutili

et al. 2004

Clin Vaccine Immunol

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The gene encoding the major envelope glycoprotein (gp51) with its signal sequence, represented by an additional NH 2 -terminal 33-residue amino acid sequence of bovine leukemia virus (BLV), was inserted into a baculovirus transfer vector. A recombinant virus expressing a secreted gp51 protein in insect cells was isolated. The recombinant gp51 expressed was characterized by using an anti-BLV monoclonal antibody by both Western blotting analysis and enzyme-linked immunosorbent assay (ELISA). The secreted gp51 was used as an antigen, and an ELISA with recombinant gp51 (rgp51) was developed for the detection of BLV antibodies. This new procedure was compared with a previous ELISA method for the detection of BLV antibodies and an agar gel immunodiffusion test performed with an unpurified BLV antigen preparation. The comparative testing of field samples showed that the ELISA with rgp51 is more specific and also suitable for the testing of pooled sera.Bovine leukemia virus (BLV), the etiological agent of enzootic bovine leukosis (EBL), is a C-type retrovirus of cattle with a worldwide distribution (5). The virus induces a persistent lymphocytosis and in some cases lymphoid tumors in domestic cattle.The BLV envelope (Env) glycoprotein, which consists of the gp51 outer membrane glycoprotein and the gp30 transmembrane glycoprotein, is directly involved in infectivity events and, like the p24 major structural protein, can elicit a strong immune response in infected cattle (23). Serological diagnosis of EBL is mainly based on screening of field samples for gp51 antibodies. It follows that diagnostic procedures that use native gp51 constitute a prerequisite for the design of an efficient EBL eradication program. In recent decades different serological methods, such as the agar gel immunodiffusion test (AGID) and the enzyme-linked immunosorbent assay (ELISA), have been developed. ELISAs are based on the use of partially purified BLV gp51 and monoclonal antibodies (MAbs) against BLV gp51 epitopes (4, 17). These procedures are particularly useful for samples with low antibody titers, such as milk samples or pooled sera. A permanent fetal lamb kidney (FLK) cell line chronically infected with BLV (FLK-BLV) is used to produce gp51. Although highly productive BLV-infected cell lines are available, their production is rather laborious, expensive, and time-consuming.In recent decades different investigators have described the expression of the BLV Env glycoproteins in heterologous expression systems such as Escherichia coli (2, 21, 22), Saccharomyces cerevisiae (11), and recombinant vaccinia virus (10, 18) systems and, recently, a baculovirus system (9, 19). However, the use of recombinant gp51 (rgp51) in an ELISA method has not yet been described. The aim of this paper is therefore to describe the production of secreted rgp51 by recombinant baculovirus in insect cells and its use in an ELISA for the detection of BLV antibodies. MATERIALS AND METHODS Cells and viruses.BLV-infected and uninfected FLK cells (National Veterinary Laborat...

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Section: Discussionsupporting

confidence: 70%

Section: Discussionmentioning

confidence: 89%

mentioning

confidence: 99%

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Expression of the Bovine Leukemia Virus Envelope Glycoprotein (gp51) by Recombinant Baculovirus and Its Use in an Enzyme-Linked Immunosorbent Assay

Giuseppe

Feliziani

Rutili

et al. 2004

Clin Vaccine Immunol

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“…The N-linked glycans present on rG1-G2 are likely to comprise high mannose type (Man 9 -5 GlcNAc 2 ) or short truncated structures (Man 3-2 GlcNAc 2 ) since these are most commonly observed on proteins expressed in cells derived from S. frugiperda (64 -70) and are released by treatment with N-glycosidase F. However, we cannot exclude the possibility that the N-glycans may be of the complex type with or without terminal sialic acid residues, which are uncommonly reported for these cells (66,71). Some studies have shown that the N-linked glycans on recombinant proteins secreted from insect cells are important for functional properties.…”

Section: Discussionmentioning

confidence: 92%

Recombinant Human Aggrecan G1-G2 Exhibits Native Binding Properties and Substrate Specificity for Matrix Metalloproteinases and Aggrecanase

Mercuri

Doege

Arner

et al. 1999

Journal of Biological Chemistry

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A recombinant human aggrecan G1-G2 fragment comprising amino acids Val 1 -Arg 656 has been expressed in Sf21 cells using a baculovirus expression system. The recombinant G1-G2 (rG1-G2) was purified to homogeneity by hyaluronan-Sepharose affinity chromatography followed by high performance liquid chromatography gel filtration, and gave a single band of M r 90,000 -95,000 by silver stain or immunoblotting with monoclonal antibody 1-C-6. The expressed G1-G2 bound to both hyaluronan and link protein indicating that the immunoglobulin-fold motif and proteoglycan tandem repeat loops of the G1 domain were correctly folded. Further analysis of secondary structure by rotary shadowing electron microscopy confirmed a double globe appearance, but revealed that the rG1-G2 was more compact than its native counterpart. The size of rG1-G2 by SDS-polyacrylamide gel electorphoresis was unchanged following digestion with keratanase and keratanase II and reduced by only 2-5 kDa following digestion with either O-glycosidase or N-glycosidase F. Recombinant G1-G2 was digested with purified matrix metalloproteinases (MMP), isolated aggrecanase, purified atrolysin C, or proteinases present in conditioned medium from cartilage explant cultures, and the products analyzed on SDS gels by silver stain and immunoblotting. sequences were used to confirm aggrecanase cleavage at the Glu 373 2 Ala bond. Cleavage at the authentic MMP and aggrecanase sites revealed that these proteinases have the same specificity for rG1-G2 as for native aggrecan. Incubation of rG1-G2 with conditioned medium from porcine cartilage cultures revealed that active soluble aggrecanase but no active MMPs, was released following stimulation with interleukin-1␣ or retinoic acid. Atrolysin C, which cleaves native bovine aggrecan at both the aggrecanase and MMP sites, efficiently cleaved rG1-G2 at the aggrecanase site but failed to cleave at the MMP site. In contrast, native glycosylated G1-G2 with or without keratanase treatment was cleaved by atrolysin C at both the aggrecanase and MMP sites. The results suggest that the presence or absence per se of keratan sulfate on native G1-G2 does not affect the activity of atrolysin C toward the two sites.Aggrecan is a chondroitin sulfate and keratan sulfate-bearing proteoglycan. It is present in cartilage as large multimolecular aggregates bound noncovalently to hyaluronan (HA). 1Aggregate formation is mediated via the proteoglycan tandem repeat loops in the N-terminal G1 domain which bind to decasaccharide units within the polymeric HA. This binding is further stabilized by link protein, which binds to form stable trimeric complexes. The formation of large aggregate structures provides a mechanism for trapping aggrecan in the tissue and preventing its loss by diffusion. A major feature of the pathology of arthritis is the gradual loss of aggrecan from the cartilage matrix. This loss involves proteolysis of the core protein and release into culture medium (1, 2) or synovial fluid (3) of large fragments lacking G1 domains that are t...

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“…This conclusion was weakened by the lack of suitable controls, as discussed above. Similarly, the conclusion that a bovine leukemia virus glycoprotein expressed by recombinant baculovirus-infected insect cells contains sialic acid was weakened by the fact that it was based on the use of WGA (wheat germ agglutinin), which interacts poorly with sialyl residues and has other specificities (Russo et al, 1998).…”

Section: Sialylation Of Recombinant Glycoproteins In Baculovirus-lnsementioning

confidence: 99%

Glycoproteins from Insect Cells: Sialylated or Not?

Marchal

Jarvis²,

Cacan³

et al. 2001

Biological Chemistry

159

122

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Our growing comprehension of the biological roles of glycan moieties has created a clear need for expression systems that can produce mammalian-type glycoproteins. In turn, this has intensified interest in understanding the protein glycosylation pathways of the heterologous hosts that are commonly used for recombinant glycoprotein expression. Among these, insect cells are the most widely used and, particularly in their role as hosts for baculovirus expression vectors, provide a powerful tool for biotechnology. Various studies of the glycosylation patterns of endogenous and recombinant glycoproteins produced by insect cells have revealed a large variety of O-and Nlinked glycan structures and have established that the major processed O-and N-glycan species found on these glycoproteins are (Galβ1,3)GalNAc-O-Ser/Thr and Man3(Fuc)GlcNAc2-N-Asn, respectively. However, the ability or inability of insect cells to synthesize and compartmentalize sialic acids and to produce sialylated glycans remains controversial. This is an important issue because terminal sialic acid residues play diverse biological roles in many glyco-conjugates. While most work indicates that insect cell-derived glycoproteins are not sialylated, some wellcontrolled studies suggest that sialylation can occur. In evaluating this work, it is important to recognize that oligosaccharide structural determination is tedious work, due to the infinite diversity of this class of compounds. Furthermore, there is no universal method of glycan analysis; rather, various strategies and techniques can be used, which provide gly-cobiologists with relatively more or less precise and reliable results. Therefore, it is important to consider the methodology used to assess glycan structures when evaluating these studies. The purpose of this review is to survey the studies that have contributed to our current view of glycoprotein sialylation in insect cell systems, according to the methods used. Possible reasons for the disagreement on this topic in the literature, which include the diverse origins of biological material and experimental artifacts, will be discussed. In the final analysis, it appears that if insect cells have the genetic potential to perform sialylation of glycoproteins, this is a highly specialized function that probably occurs rarely. Thus, the production of sialylated recombinant glycoproteins in the baculovirus-insect cell system will require metabolic engineering efforts to extend the native protein glycosylation pathways of insect cells.

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Expression of bovine leukemia virus ENV glycoprotein in insect cells by recombinant baculovirus

Cited by 10 publications

References 29 publications

Expression of the Bovine Leukemia Virus Envelope Glycoprotein (gp51) by Recombinant Baculovirus and Its Use in an Enzyme-Linked Immunosorbent Assay

Expression of the Bovine Leukemia Virus Envelope Glycoprotein (gp51) by Recombinant Baculovirus and Its Use in an Enzyme-Linked Immunosorbent Assay

Recombinant Human Aggrecan G1-G2 Exhibits Native Binding Properties and Substrate Specificity for Matrix Metalloproteinases and Aggrecanase

Glycoproteins from Insect Cells: Sialylated or Not?

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