Enhanced secretion from insect cells of a foreign protein fused to the honeybee melittin signal peptide

Tessier, Daniel C.; Thomas, David Y.; Khouri, Henry E.; Laliberté, France; Vernet, Thierry

doi:10.1016/0378-1119(91)90171-7

Cited by 273 publications

(173 citation statements)

References 18 publications

Supporting

Mentioning

161

Contrasting

Unclassified

Order By: Relevance

“…[31][32][33] In each of these systems, hirudin secretion was directed by heterologous signal peptides already known to function efficiently in these species. Because our goal was to express hirudin in a mammalian cell and because there is evidence that use of host cell species-specific signal peptides can result in enhanced secretion and more reliable post-translational processing, 34,35 we initially used the human t-PA signal peptide to direct hirudin secretion from human endothelial cells. The t-PA signal peptide functions well in human endothelial cells 36 and has also been used successfully as a heterologous signal peptide in other systems.…”

Section: Discussionmentioning

confidence: 99%

Retroviral vector-mediated expression of hirudin by human vascular endothelial cells: implications for the design of retroviral vectors expressing biologically active proteins

et al. 1999

View full text Add to dashboard Cite

We constructed a hirudin cDNA cassette, HV-1.1, that and mass spectroscopic analysis revealed the presence of encodes mature hirudin variant-1 fused to the signal pepan extra N-terminal serine residue, indicating aberrant tide of human tissue-type plasminogen activator (t-PA).cleavage of the t-PA signal peptide and likely accounting The cassette was subcloned into retroviral vectors and for the diminished activity. We therefore constructed a used to transduce human vascular endothelial cells in vitro.second cDNA cassette, HV-1.2, in which hirudin secretion Hirudin antigen and activity were measured by ELISA and was directed by the signal peptide of human growth horthrombin inhibition assays, respectively. Transduced cells mone. Hirudin expressed from the HV-1.2 cassette had a secreted up to 35 ± 2 ng/10 6 cells/24 h of biologically active specific activity of 13.5 ± 0.2 ATU/ g. Protein sequencing hirudin; expression was stable for at least 7 weeks.and mass spectroscopic analysis demonstrated proper Recombinant hirudin, expressed from the HV-1.1 cassette, cleavage of the growth hormone signal peptide. Thus, we had a specific activity of 7.1 ± 0.2 antithrombin units per achieved high level retrovirus-mediated secretion of biomicrogram (ATU/ g), compared with specific activities of logically active hirudin from endothelial cells in vitro. Use approximately 12 ATU/ g for both native leech hirudin and of these vectors may permit sustained local antagonism of recombinant hirudin produced in yeast. Protein sequencing thrombin activity in vivo.

show abstract

Section: Discussionmentioning

confidence: 99%

Retroviral vector-mediated expression of hirudin by human vascular endothelial cells: implications for the design of retroviral vectors expressing biologically active proteins

et al. 1999

View full text Add to dashboard Cite

show abstract

“…pHis 6 -Scap(Loop1) encodes, in sequential order from the NH 2 terminus, the signal sequence from honeybee mellitin (19), an epitope tag consisting of six histidines, a TEV protease cleavage site (20), and luminal Loop 1 of Scap (amino acids 46 -269) (see supplemental Fig. S1).…”

Section: Methodsmentioning

confidence: 99%

Identification of Luminal Loop 1 of Scap Protein as the Sterol Sensor That Maintains Cholesterol Homeostasis

Motamed

Zhang

Wang

et al. 2011

Journal of Biological Chemistry

View full text Add to dashboard Cite

The endoplasmic reticulum (ER)4 protein Scap is unique in nature because it serves as a cholesterol sensor that ensures the proper amount of cholesterol in membranes of animal cells (1, 2). The function of Scap derives from its ability to mediate the regulated transport of sterol regulatory element-binding proteins (SREBPs) from ER to Golgi. SREBPs are a family of three transcription factors that activate all of the genes necessary to produce cholesterol, fatty acids, and triglycerides (3). The SREBPs are synthesized as intrinsic transmembrane proteins of the ER. Immediately after their synthesis, the SREBPs bind to Scap, which serves as the nidus for incorporation into COPIIcoated vesicles, which bud from the ER and travel to the Golgi. There the SREBPs are processed sequentially by two proteases, thereby releasing the active transcriptional fragments that travel to the nucleus. When cholesterol accumulates in ER membranes, Scap binds the cholesterol and undergoes a conformational change that causes it to bind to Insig, an ER-resident protein (4). As a result of the conformational change and its stabilization by Insig (5), the Scap-SREBP complex is no longer incorporated into budding vesicles, and the active fragment cannot reach the nucleus. As a result, synthesis of cholesterol and fatty acids declines.The 1276 amino acids of Scap can be divided into two functional regions (see Fig. 1). The COOH-terminal domain of ϳ540 amino acids extends into the cytosol. It contains at least four WD repeat sequences that mediate its binding to SREBPs. The NH 2 -terminal region of ϳ735 amino acids is the membrane attachment domain. It contains eight ␣-helices separated by hydrophilic loops (6). Three of the loops (Loops 1, 6, and 7) are long enough to have significant structure. Helices 2-6 contain the Insig binding site (7,8). Loop 6, which faces the cytosol, contains the hexapeptide sequence MELADL, which serves as the binding site for the COPII proteins that cluster the Scap-SREBP complex into COPII-coated vesicles that bud from ER membranes (2, 9). When the cholesterol content of ER membranes exceeds a sharp threshold of 4 -5% of total lipids, the cholesterol binds to the membrane region of Scap (10), and this elicits a conformational change in Loop 6 that can be monitored by a protease protection assay (11). The change is reflected by the exposure of a novel arginine (Arg 505 ) to cleavage by trypsin (Fig. 1).The cholesterol-induced conformational change in Loop 6 causes the MELADL sequence to become inaccessible to COPII proteins, thereby precluding transport to the Golgi (2). Although the conformational change does not require Insig, binding to Insig stabilizes the inactive conformation, thereby lowering the threshold for cholesterol (10).Our previous cholesterol-binding studies were performed with a recombinant form of Scap that contained the entire membrane attachment domain (TM1-8) (1,12). Within this domain, the precise site of cholesterol binding was not established. In the current study, we localize the choleste...

show abstract

“…The upstream primer was 5Ј-CGGATCCATACGTAGATGGCGGTCTGTGG-GA-3Ј containing the N-terminal end of the disintegrin-like domain, corresponding to nucleotides 1162-1180 of GenBank TM Accession no. U16242 plus upstream BamHI and SnaBI restriction sites for in-frame insertion (using the BamHI site) into the baculovirus expression vector pVT-Bac (24), which contains the melittin signal sequence. In the first step, an hemagglutinin (HA) epitope tag was added at the C-terminal end using the following primer: 5Ј-CGGGATCCTAGGCATAATCTGG-CACATCATAAGGGTACTGGTGACCATCATAGCCCAAGAGTGTC-3Ј; this primer corresponds to nucleotides 1834 -1855 of the mouse fertilin ␤ cDNA (Accession no.…”

Section: Methodsmentioning

confidence: 99%

Sequence-specific Interaction between the Disintegrin Domain of Mouse ADAM 2 (Fertilin β) and Murine Eggs

Bigler¹,

Takahashi²,

Chen³

et al. 2000

Journal of Biological Chemistry

105

View full text Add to dashboard Cite

Little is yet known about the biological and biochemical properties of the disintegrin-like domains of ADAM (a disintegrin and metalloprotease) proteins. Mouse ADAM 2 (mADAM 2; fertilin ␤) is a sperm surface protein involved in murine fertilization. We produced recombinant proteins containing the disintegrin-like domain of mADAM 2 in both insect cells and in bacteria. The protein produced in insect cells (baculo D؉C) contained a signal sequence followed by the disintegrin-like and cysteine-rich domains; it was purified from the medium of recombinant baculovirus-infected cells. A bacterial construct containing the disintegrin-like domain was produced in Escherichia coli as a glutathione S-transferase chimera. Baculo D؉C, as well as the D domain of the bacterial construct (released with thrombin), bound to the microvillar surface of murine eggs. Using concentrations in the range of 1 to 5 M, both recombinant proteins strongly inhibited sperm-egg binding and fusion; the baculovirus-produced protein exhibited a somewhat greater extent of inhibition (ϳ75 versus ϳ55% maximal inhibition). Substitution of alanine for each of the five charged residues within the disintegrin loop of mADAM 2 revealed a critical importance for the aspartic acid at position nine. Binding of both recombinant proteins to the egg was inhibited by the function blocking anti-␣ 6 monoclonal antibody, GoH3, but not by a nonfunctionblocking anti-␣ 6 monoclonal antibody. Binding was also inhibited by a peptide analogue of, and with an antibody against, the disintegrin loop of mADAM 2. ADAMs1 are a large group of type I integral membrane glycoproteins that contain a disintegrin and a metalloprotease domain (1, 2). Following their metalloprotease and disintegrinlike domains, they contain a cysteine-rich domain, an EGF repeat, a transmembrane domain, and a cytoplasmic tail. Their closest relatives are the P-III snake venom metalloproteases (SVMPs), which are secreted proteins that contain a disintegrin-like and a metalloprotease domain as well as a cysteinerich domain. P-II SVMPs contain metalloprotease and disintegrin domains but lack the cysteine-rich (and other) domains.The disintegrin domains of the P-II SVMPs have a 13-amino acid loop containing, at their tips, sequences such as RGD (kistrin and echistatin), KGD (barbourin), and MVD (atrolysin E). Several P-II snake disintegrins have been shown to bind to the platelet integrin, ␣ IIb ␤ 3, thereby preventing binding of fibrinogen and inhibiting platelet aggregation. Inhibition of platelet aggregation accounts, in part, for the severe hemorrhagic response in snakebite victims. The disintegrin-like domains of P-III SVMPs differ from their P-II counterparts in having two additional cysteine residues and a 14-residue predicted loop that aligns with the 13-amino acid "RGD" loop found in the P-II disintegrins. One of the additional cysteine residues is near the center of the disintegrin loop. The disintegrin-like domain of the P-III SVMP atrolysin A has been characterized. Atrolysin A purified from snake ...

show abstract

Enhanced secretion from insect cells of a foreign protein fused to the honeybee melittin signal peptide

Cited by 273 publications

References 18 publications

Retroviral vector-mediated expression of hirudin by human vascular endothelial cells: implications for the design of retroviral vectors expressing biologically active proteins

Retroviral vector-mediated expression of hirudin by human vascular endothelial cells: implications for the design of retroviral vectors expressing biologically active proteins

Identification of Luminal Loop 1 of Scap Protein as the Sterol Sensor That Maintains Cholesterol Homeostasis

Sequence-specific Interaction between the Disintegrin Domain of Mouse ADAM 2 (Fertilin β) and Murine Eggs

Contact Info

Product

Resources

About