scholarly journals 15 N labeling method of peptides using a thioredoxin gene fusion expression system: an application to ACTH-(1-24)

FEBS Letters ◽  
1996 ◽  
Vol 379 (1) ◽  
pp. 47-50 ◽  
Author(s):  
Koichi Uegaki ◽  
Nobuaki Nemoto ◽  
Masato Shimizu ◽  
Takashi Wada ◽  
Yoshimasa Kyogoku ◽  
...  
Keyword(s):  
Gene Fusion ◽  
Expression System ◽  
Fusion Expression ◽  
Labeling Method

A Thioredoxin Gene Fusion Expression System That Circumvents Inclusion Body Formation in the E. coli Cytoplasm

1993 ◽  
Vol 11 (2) ◽  
pp. 187-193 ◽  
Author(s):  
Edward R. LaVallie ◽  
Elizabeth A. DiBlasio ◽  
Sharlotte Kovacic ◽  
Kathleen L. Grant ◽  
Paul F. Schendel ◽  
...  
Keyword(s):  
Inclusion Body ◽  
Gene Fusion ◽  
Expression System ◽  
Fusion Expression ◽  
Body Formation ◽  
E Coli ◽  
Inclusion Body Formation

scholarly journals Expanding the molecular toolbox for Lactococcus lactis: construction of an inducible thioredoxin gene fusion expression system

2011 ◽  
Vol 10 (1) ◽  
pp. 66 ◽  
Author(s):  
François P Douillard ◽  
Mary O'Connell-Motherway ◽  
Christian Cambillau ◽  
Douwe van Sinderen
Keyword(s):  
Lactococcus Lactis ◽  
Gene Fusion ◽  
Expression System ◽  
Fusion Expression

scholarly journals A Protein Disulfide Isomerase Gene Fusion Expression System That Increases the Extracellular Productivity ofBacillus brevis

2000 ◽  
Vol 66 (2) ◽  
pp. 638-642 ◽  
Author(s):  
Tsutomu Kajino ◽  
Chikara Ohto ◽  
Masayoshi Muramatsu ◽  
Shusei Obata ◽  
Shigezo Udaka ◽  
...  
Keyword(s):  
Protein Disulfide Isomerase ◽  
Gene Fusion ◽  
Expression System ◽  
Biologically Active ◽  
Fusion Expression ◽  
Fusion Partner ◽  
Heterologous Proteins ◽  
Bacillus Brevis ◽  
Disulfide Isomerase ◽  
Protein Disulfide

ABSTRACT We have developed a versatile Bacillus brevisexpression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system.

Phosphatidylinositol 3-kinase activation is mediated by high-affinity interactions between distinct domains within the p110 and p85 subunits

1994 ◽  
Vol 14 (1) ◽  
pp. 42-49
Author(s):  
K H Holt ◽  
L Olson ◽  
W S Moye-Rowley ◽  
J E Pessin
Keyword(s):  
Gene Fusion ◽  
Kinase Activity ◽  
Expression System ◽  
Sh2 Domain ◽  
Full Length ◽  
Transactivation Domain ◽  
Phosphatidylinositol 3 Kinase ◽  
Sh2 Domains ◽  
Amino Terminal ◽  
Phosphatidylinositol 3

Domains of interaction between the p85 and p110 subunits of phosphatidylinositol 3-kinase (PI 3-kinase) were studied with the yeast two-hybrid expression system. A gene fusion between the GAL4 transactivation domain and p85 activated transcription from a GAL1-lacZ reporter gene when complemented with a gene fusion between the GAL4 DNA binding domain and p110. To define subdomains responsible for this interaction, a series of p85 deletion mutants were analyzed. A 192-amino-acid inter-SH2 (IS) fragment (residues 429 to 621) was the smallest determinant identified that specifically associated with p110. In analogous experiments, the subdomain within p110 responsible for interaction with p85 was localized to an EcoRI fragment encoding the amino-terminal 127 residues. Expression of these two subdomains [p85(IS) with p110RI] resulted in 100-fold greater reporter activity than that obtained with full-length p85 and p110. Although the p85(IS) domain conferred a strong interaction with the p110 catalytic subunit, this region was not sufficient to impart phosphotyrosine peptide stimulation of PI 3-kinase activity. In contrast, coexpression of the p110 subunit with full-length p85 or with constructs containing the IS sequences flanked by both SH2 domains of p85 [p85(n/cSH2)] or either of the individual SH2 domains [p85(nSH2+IS) or p85(IS+cSH2)] resulted in PI 3-kinase activity that was activated by a phosphotyrosine peptide. These data suggest that phosphotyrosine peptide binding to either SH2 domain generates an intramolecular signal propagated through the IS region to allosterically activate p110.

Arabidopsis thaliana and Saccharomyces cerevisiae NHX1 genes encode amiloride sensitive electroneutral Na+/H+ exchangers

2000 ◽  
Vol 351 (1) ◽  
pp. 241-249 ◽  
Author(s):  
Catherine P. DARLEY ◽  
Olivier C. M. VAN WUYTSWINKEL ◽  
Karel VAN DER WOUDE ◽  
Willem H. MAGER ◽  
Albertus H. DE BOER
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Fusion ◽  
Direct Evidence ◽  
Protein Targeting ◽  
Expression System ◽  
Yeast Cells ◽  
Null Mutant ◽  
Heterologous Expression System ◽  
Na Ions ◽  
Convenient Model

Sodium at high millimolar levels in the cytoplasm is toxic to plant and yeast cells. Sequestration of Na+ ions into the vacuole is one mechanism to confer Na+-tolerance on these organisms. In the present study we provide direct evidence that the ArabidopsisthalianaAt-NHX1 gene and the yeast NHX1 gene encode low-affinity electroneutral Na+/H+ exchangers. We took advantage of the ability of heterologously expressed At-NHX1 to functionally complement the yeast nhx1-null mutant. Experiments on vacuolar vesicles isolated from yeast expressing At-NHX1 or NHX1 provided direct evidence for pH-gradient-energized Na+ accumulation into the vacuole. A major difference between NHX1 and At-NHX1 is the presence of a cleavable N-terminal signal peptide (SP) in the former gene. Fusion of the SP to At-NHX1 resulted in an increase in the magnitude of Na+/H+ exchange, indicating a role for the SP in protein targeting or regulation. Another distinguishing feature between the plant and yeast antiporters is their sensitivity to the diuretic compound amiloride. Whereas At-NHX1 was completely inhibited by amiloride, NHX1 activity was reduced by only 20–40%. These results show that yeast as a heterologous expression system provides a convenient model to analyse structural and regulatory features of plant Na+/H+ antiporters.

scholarly journals Phosphatidylinositol 3-kinase activation is mediated by high-affinity interactions between distinct domains within the p110 and p85 subunits.

1994 ◽  
Vol 14 (1) ◽  
pp. 42-49 ◽  
Author(s):  
K H Holt ◽  
L Olson ◽  
W S Moye-Rowley ◽  
J E Pessin
Keyword(s):  
Gene Fusion ◽  
Kinase Activity ◽  
Expression System ◽  
Sh2 Domain ◽  
Full Length ◽  
Transactivation Domain ◽  
Phosphatidylinositol 3 Kinase ◽  
Sh2 Domains ◽  
Amino Terminal ◽  
Phosphatidylinositol 3

Domains of interaction between the p85 and p110 subunits of phosphatidylinositol 3-kinase (PI 3-kinase) were studied with the yeast two-hybrid expression system. A gene fusion between the GAL4 transactivation domain and p85 activated transcription from a GAL1-lacZ reporter gene when complemented with a gene fusion between the GAL4 DNA binding domain and p110. To define subdomains responsible for this interaction, a series of p85 deletion mutants were analyzed. A 192-amino-acid inter-SH2 (IS) fragment (residues 429 to 621) was the smallest determinant identified that specifically associated with p110. In analogous experiments, the subdomain within p110 responsible for interaction with p85 was localized to an EcoRI fragment encoding the amino-terminal 127 residues. Expression of these two subdomains [p85(IS) with p110RI] resulted in 100-fold greater reporter activity than that obtained with full-length p85 and p110. Although the p85(IS) domain conferred a strong interaction with the p110 catalytic subunit, this region was not sufficient to impart phosphotyrosine peptide stimulation of PI 3-kinase activity. In contrast, coexpression of the p110 subunit with full-length p85 or with constructs containing the IS sequences flanked by both SH2 domains of p85 [p85(n/cSH2)] or either of the individual SH2 domains [p85(nSH2+IS) or p85(IS+cSH2)] resulted in PI 3-kinase activity that was activated by a phosphotyrosine peptide. These data suggest that phosphotyrosine peptide binding to either SH2 domain generates an intramolecular signal propagated through the IS region to allosterically activate p110.

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