Site-directed mutagenesis of human lysyl hydroxylase expressed in insect cells

1996 ◽  
Vol 15 (3) ◽  
pp. 196
Author(s):  
A. Pirskanen ◽  
A.-M. Kaimio ◽  
R. Myllylä ◽  
K.I. Kivirikko
Keyword(s):  
Insect Cells ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Lysyl Hydroxylase

scholarly journals Site-directed Mutagenesis of Human Lysyl Hydroxylase Expressed in Insect Cells

1996 ◽  
Vol 271 (16) ◽  
pp. 9398-9402 ◽  
Author(s):  
Asta Pirskanen ◽  
Anne-Maarit Kaimio ◽  
Raili Myllylä ◽  
Kari I. Kivirikko
Keyword(s):  
Insect Cells ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Lysyl Hydroxylase

scholarly journals Expression and site-directed mutagenesis of mouse prostaglandin E2 receptor EP3 subtype in insect cells

1995 ◽  
Vol 307 (2) ◽  
pp. 493-498 ◽  
Author(s):  
C Huang ◽  
H H Tai
Keyword(s):  
Ligand Binding ◽  
Prostaglandin E2 ◽  
Insect Cells ◽  
Expression System ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Binding Studies ◽  
Comparable Amount ◽  
Ep3 Receptor

A cDNA encoding for mouse prostaglandin E2 (PGE2) receptor EP3 subtype was cloned from a mouse kidney cDNA library by PCR using terminal primers derived from the known sequence of mouse lung EP3 receptor cDNA. The cloned cDNA was confirmed by sequencing and was expressed in Trichoplusia ni (MG1) insect cells using a baculovirus expression system. A specific protein of 60 kDa was detected by immunoblot with antibodies generated against a unique decapeptide sequence present in the second extracellular loop of the EP3 receptor. Specific binding of [3H]PGE2 with a Kd of 3 nM was also found in the membrane fraction of the insect cells. Ligand binding of the receptor was further studied by site-directed mutagenesis. Arg-309 of the receptor was separately mutated to lysine, glutamate and valine. cDNAs of the wild-type and mutant EP3 receptors were respectively expressed and studied in MG1 insect cells. Binding studies indicated that both glutamate and valine mutant EP3 receptors had no binding of [3H]PGE2. On the contrary, the lysine mutant receptor exhibited an even tighter binding (Kd = 1.3 nM) than the wild-type EP3 receptor. Immunoblot studies indicated that these receptors were expressed in a comparable amount in MG1 insect cells. These results suggest that Arg-309 of EP3 receptor may be essential in ligand binding through ionic interaction.

scholarly journals Expression of two different forms of cDNA for thromboxane synthase in insect cells and site-directed mutagenesis of a critical cysteine residue

1993 ◽  
Vol 295 (2) ◽  
pp. 457-461 ◽  
Author(s):  
Z Xia ◽  
R F Shen ◽  
S J Baek ◽  
H H Tai
Keyword(s):  
Cdna Library ◽  
Insect Cells ◽  
Specific Activity ◽  
Short Form ◽  
Expression System ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Mutant Enzyme ◽  
Directed Mutagenesis ◽  
Thromboxane Synthase

cDNA coding for human placental thromboxane synthase (EC 5.3.99.5) was amplified by PCR from a human placental cDNA library and sequenced. This cDNA and a shorter cDNA isolated from a human lung cDNA library with a deletion of 163 bp near the 3′ end were expressed in Spodoptera frugiperda (Sf9) insect cells using a baculovirus expression system. The cDNA from human placenta was expressed as an active enzyme (60 kDa) with a specific activity higher than those reported from other cell types, whereas the shorter cDNA was expressed in an inactive form (52 kDa). The active recombinant enzyme appeared to be unglycosylated as the molecular mass and the enzyme activity were not altered in the presence of tunicamycin. Site-directed mutagenesis was performed to convert a cysteine at position 480 in thromboxane synthase to a serine. This cysteine is found to be highly conserved in related cytochrome P-450 enzymes. The mutant enzyme was found to be inactive, although Western blot, immunoprecipitation and SDS/PAGE analysis indicated that the mutant enzyme was expressed at a level comparable with the wild-type enzyme. These results suggest that Cys-480 is essential for the enzyme catalytic activity and that the short-form cDNA may be a non-functional transcript.

scholarly journals Analysis of individual azurocidin N-glycosylation sites in regard to its secretion by insect cells, susceptibility to proteolysis and antibacterial activity.

2007 ◽  
Vol 54 (3) ◽  
pp. 567-573 ◽  
Author(s):  
Katarzyna Indyk ◽  
Teresa Olczak ◽  
Justyna Ciuraszkiewicz ◽  
Wiesław Watorek ◽  
Mariusz Olczak
Keyword(s):  
Antibacterial Activity ◽  
Neutrophil Elastase ◽  
Insect Cells ◽  
Sequence Similarity ◽  
Cathepsin G ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Proteinase 3 ◽  
Primary Sequence ◽  
Glycosylation Sites

Azurocidin is an inactive serine protease homolog with primary sequence similarity to neutrophil elastase, cathepsin G, and proteinase 3. The aim of this study was to investigate possible consequences of differential glycosylation of azurocidin in regard to its secretion, protein stability as measured by susceptibility to proteolysis, and antibacterial activity. Site-directed mutagenesis was employed to generate mutant azurocidin variants lacking individual N-glycosylation sites. Our results show that N-linked glycans may play a role in proper azurocidin folding and subsequent secretion by insect cells. We also demonstrate that N-linked glycosylation contributes to azurocidin stability by protecting it from proteolysis. The lack of N-glycosylation at individual sites does not significantly influence the azurocidin antibacterial activity.

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