Expression of the human EGF receptor with ligand-stimulatable kinase activity in insect cells using a baculovirus vector.

Greenfield, C.; Patel, G. H.; Clark, Stella; Jones, Nic; Waterfield, Mike

doi:10.1002/j.1460-2075.1988.tb02793.x

Cited by 112 publications

(59 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…However, although SRK was correctly inserted into the membrane, with the C terminus extending into the cytoplasm, the majority of the recombinant SRK was not targeted to the plasmalemma of the insect cells. This finding contrasts with results obtained for animal receptor kinases, such as epidermal growth factor receptor or insulin receptor, which have been shown to be correctly addressed to the plasmalemma when expressed in insect cells (34,35). Nevertheless, it is worth noting that plant K ϩ channels, which are plasma membrane proteins, also were retained mainly in the internal cellular membranes when expressed in the insect cell͞baculo-virus system (36,37).…”

Section: Discussioncontrasting

confidence: 60%

The integral membrane S-locus receptor kinase of Brassica has serine/threonine kinase activity in a membranous environment and spontaneously forms oligomers in planta

Giranton¹

2000

Proceedings of the National Academy of Sciences

View full text Add to dashboard Cite

To gain further insight into the mode of action of S-locus receptor kinase (SRK), a receptor-like kinase involved in the self-incompatibility response in Brassica, different recombinant SRK proteins have been expressed in a membranous environment using the insect cell͞baculovirus system. Recombinant SRK proteins exhibited properties close to those of the endogenous stigmatic SRK protein and were found to autophosphorylate on serine and threonine residues in insect cell microsomes. Autophosphorylation was constitutive because it did not require the presence of pollen or stigma extracts in the phosphorylation buffer. Phosphorylation was shown to occur in trans, suggesting the existence of constitutive homooligomers of membrane-anchored recombinant SRK. To investigate the physiological relevance of these results, we have examined the oligomeric status of SRK in planta in cross-linking experiments and by velocity sedimentation on sucrose gradients. Our data strongly suggest that SRK is associated both with other SRK molecules and other stigma proteins in nonpollinated flowers. These findings may have important implications for our understanding of self-pollen signaling. baculovirus ͉ oligomerization ͉ sporophytic self-incompatibility I n the Brassica family, the cell-cell interaction that leads to the rejection of self-pollen at the stigmatic surface [the selfincompatibility (SI) reaction] is controlled genetically by the multiallelic S-locus (1). When the pollen parent and the stigma share common S haplotypes, pollen germination or pollen tube growth is inhibited, thereby preventing self-fertilization. Several genes have been localized at the S locus but only two, the S-locus glycoprotein (SLG) and the S-locus receptor kinase (SRK) genes, exhibit the polymorphic nature expected for genes involved in the SI response (2, 3). SLG encodes a secreted glycoprotein that accumulates in the cell wall of papillae (4), whereas SRK encodes a plasmalemma-anchored glycoprotein (5, 6). SRK is structurally analogous to animal receptor kinases and belongs to the plant receptor-like kinase (RLK) family (7). Although its membrane topology has not been experimentally defined, DNA sequence analysis predicts that SRK consists of three domains: an extracellular glycosylated N-terminal domain (the S-domain) that shares extensive homology with SLG, a membrane-spanning domain, and a cytoplasmic domain (3). The cytoplasmic domain has been shown to have serine͞threonine kinase activity when expressed in bacteria (8). In the S 3 haplotype, SRK has been shown to encode, in addition to the integral membrane SRK protein, a soluble truncated form corresponding to the S-domain of SRK, the eSRK protein (9).Although there is some controversy as to whether SLG is required for the SI response (10, 11), there is strong evidence that SRK is necessary. For example, SRK was shown to be mutated in two Brassica lines that exhibited a self-compatible phenotype (12, 13). Moreover, Stahl and coworkers (14) recently have shown that the SI phenotype was altered i...

show abstract

Section: Discussioncontrasting

confidence: 60%

The integral membrane S-locus receptor kinase of Brassica has serine/threonine kinase activity in a membranous environment and spontaneously forms oligomers in planta

Giranton¹

2000

Proceedings of the National Academy of Sciences

View full text Add to dashboard Cite

show abstract

“…Proteins in medium (supplemented with 10% fetal calf serum) or cell lysates prepared as described [21] were separated on a 15% SDS/polyacrylamide gel. Molecular mass markers were stained with Coomassie brilliant blue R. Electrophoretic transfer of proteins was performed essentially according to the method of Burnette [22].…”

Section: Western Blotsmentioning

confidence: 99%

Structure of preproattacin and its processing in insect cells infected with a recombinant baculovirus

Gunne¹,

Hellers²,

Steiner³

1990

European Journal of Biochemistry

View full text Add to dashboard Cite

From a cDNA library made from fatbody of immunized Hyalophora cecropia pupae, a full-length clone corresponding to basic attacin was isolated. The corresponding amino acid sequence suggests that basic attacin is synthesized as a 233-residue preproprotein. This was confirmed by cloning the cDNA fragment encoding the basic attacin in the baculovirus Autographa californica nuclear polyhedrosis virus downstream of the polyhedrin promoter and expressing the protein in Spodoptera frug@erda (Sf9) cells. Biologically active attacin was also produced in last instar larvae of Trichoplusia ni after injection of recombinant virus. The concentration obtained was 300 ~ SO0 times that obtained in cell culture supernatants. In cell culture the induction kinetics of attacin were followed at both transcriptional and translational levels. From the protein processing pattern it was suggested that a protease produced by the Spodoptera cells cleaves attacin at the double arginine residues at positions 45 and 46. The instability of the attacin proteins is rationalized in terms of their random-coil structure which was deduced from circular dichroism measurements.

show abstract

“…Several studies have reported that oligosaccharide structures synthesized by insect cells differ from those synthesized by mammalian cells (Butters & Hughes, 1981 ;Hsieh & Robbins, 1984;Weber et al, 1986;Wojchowski et al, 1987;Greenfield et al, 1988;Kuroda et al, 1990;Wathen et al, 1991). In particular, insect cells appear not to process highmannose precursor oligosaccharides into mammaliantype complex structures.…”

Section: Introductionmentioning

confidence: 99%

Comparison of soluble and secreted forms of human parainfluenza virus type 3 glycoproteins expressed from mammalian and insect cells as subunit vaccines

Lehman¹,

Roof²,

Brideau³

et al. 1993

Journal of General Virology

View full text Add to dashboard Cite

Human parainfluenza virus type 3 (PIV-3) is one of the leading causes of paediatric viral respiratory disease. The PIV-3 genome encodes two envelope glycoproteins, F and HN, which are the major targets for the host antibody response. We have expressed secreted forms of the F and HN proteins and a novel chimeric FHN glycoprotein in insect cells using recombinant baculovirus vectors and secreted forms of the F and FHN glycoproteins in stably transformed Chinese hamster ovary (CHO) ceils. Comparison of the mammalian cell-and insect cell-expressed F and FHN proteins by SDS-PAGE showed that the CHO cellexpressed proteins are several kilodaltons larger in size than the baculovirus-produced proteins. A partial characterization of the oligosaccharide structures of the F and FHN proteins revealed that the size difference is due to the different oligosaccharide structures added to these proteins by the two cell lines. The F, HN and FHN proteins were immunoaffinity-purified from the culture medium of baculovirus-infected Sf9 cells and the F and FHN proteins were immunoafiinity-purified from the culture medium of CHO cells. A comparison of the immunogenicity and efficacy of the mammalian cell-and insect cell-produced FHN proteins was tested in cotton rats. The CHO cell-and baculovirus-produced FHN proteins were found to induce similar levels of PIV-3-specific ELISA-positive and neutralizing antibodies and both proteins provided near complete protection when animals were vaccinated with low doses of the FHN protein.

show abstract

Expression of the human EGF receptor with ligand-stimulatable kinase activity in insect cells using a baculovirus vector.

Cited by 112 publications

References 40 publications

The integral membrane S-locus receptor kinase of Brassica has serine/threonine kinase activity in a membranous environment and spontaneously forms oligomers in planta

The integral membrane S-locus receptor kinase of Brassica has serine/threonine kinase activity in a membranous environment and spontaneously forms oligomers in planta

Structure of preproattacin and its processing in insect cells infected with a recombinant baculovirus

Comparison of soluble and secreted forms of human parainfluenza virus type 3 glycoproteins expressed from mammalian and insect cells as subunit vaccines

Contact Info

Product

Resources

About