scholarly journals Construction of an Expression System for Site-Directed Mutagenesis of the Lantibiotic Mersacidin

2003 ◽  
Vol 69 (7) ◽  
pp. 3777-3783 ◽  
Author(s):  
Christiane Szekat ◽  
Ralph W. Jack ◽  
Dirk Skutlarek ◽  
Harald Färber ◽  
Gabriele Bierbaum
Keyword(s):  
Biological Activity ◽  
Expression System ◽  
Biosynthetic Gene Cluster ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Lipid Ii ◽  
Type Gene ◽  
Self Protection ◽  
Acid Function ◽  
Reduced Activity

ABSTRACT The lantibiotic (i.e., lanthionine-containing antibiotic) mersacidin is an antimicrobial peptide of 20 amino acids which is produced by Bacillus sp. strain HIL Y-85,54728. Mersacidin inhibits bacterial cell wall biosynthesis by binding to the precursor molecule lipid II. The structural gene of mersacidin (mrsA) and the genes for the enzymes of the biosynthesis pathway, dedicated transporters, producer self-protection proteins, and regulatory factors are organized in a biosynthetic gene cluster. For site-directed mutagenesis of lantibiotics, the engineered genes must be expressed in an expression system that contains all of the factors necessary for biosynthesis, export, and producer self-protection. In order to express engineered mersacidin peptides, a system in which the engineered gene replaces the wild-type gene on the chromosome was constructed. To test the expression system, three mutants were constructed. In S16I mersacidin, the didehydroalanine residue (Dha) at position 16 was replaced with the Ile residue found in the closely related lantibiotic actagardine. S16I mersacidin was produced only in small amounts. The purified peptide had markedly reduced antimicrobial activity, indicating an essential role for Dha16 in biosynthesis and biological activity of mersacidin. Similarly, Glu17, which is thought to be an essential structure in mersacidin, was exchanged for alanine. E17A mersacidin was obtained in good yields but also showed markedly reduced activity, thus confirming the importance of the carboxylic acid function at position 17 in the biological activity of mersacidin. Finally, the exchange of an aromatic for an aliphatic hydrophobic residue at position 3 resulted in the mutant peptide F3L mersacidin; this peptide showed only moderately reduced activity.

scholarly journals Biosynthesis of the v-sis gene product: signal sequence cleavage, glycosylation, and proteolytic processing.

1986 ◽  
Vol 6 (4) ◽  
pp. 1343-1348 ◽  
Author(s):  
M Hannink ◽  
D J Donoghue
Keyword(s):  
Biological Activity ◽  
Growth Factor ◽  
Gene Product ◽  
Signal Sequence ◽  
Intracellular Localization ◽  
Growth Factor Receptor ◽  
Proteolytic Processing ◽  
Site Directed Mutagenesis ◽  
Platelet Derived Growth Factor ◽  
Directed Mutagenesis

The v-sis oncogene and its cellular homolog c-sis encode chain B of platelet-derived growth factor. Cells transformed by v-sis produce a platelet-derived growth factor-related molecule which is able to stimulate the platelet-derived growth factor receptor in an autocrine fashion. Site-directed mutagenesis was used to construct several mutations which substitute charged residues for hydrophobic residues in the proposed signal sequence of the v-sis gene product. Two of these mutations resulted in the synthesis of altered v-sis gene products with an unexpected nuclear location and a loss of biological activity. We also report here the intracellular localization of the v-sis gene product to the endoplasmic reticulum-Golgi compartment, where signal sequence cleavage and N-linked glycosylation occur. The v-sis gene product contains no transmembrane regions, as it is completely protected within isolated microsomes from trypsin proteolysis. Site-directed mutagenesis was also used to alter a proposed proteolytic processing site in the v-sis gene product. This mutant v-sis gene, which encodes Asn-Ser in place of Lys-Arg at residues 110 to 111, was found to retain full biological activity.

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 Role of the Single Regulator MrsR1 and the Two-Component System MrsR2/K2 in the Regulation of Mersacidin Production and Immunity

2002 ◽  
Vol 68 (1) ◽  
pp. 106-113 ◽  
Author(s):  
André Guder ◽  
Tim Schmitter ◽  
Imke Wiedemann ◽  
Hans-Georg Sahl ◽  
Gabriele Bierbaum
Keyword(s):  
Abc Transporter ◽  
Response Regulator ◽  
Biosynthetic Gene Cluster ◽  
Open Reading Frames ◽  
Pcr Analysis ◽  
Knockout Mutant ◽  
Intact Cells ◽  
Lipid Ii ◽  
Two Component ◽  
Self Protection

ABSTRACT The lantibiotic mersacidin is an antimicrobial peptide of 20 amino acids which inhibits bacterial cell wall biosynthesis by binding to the precursor molecule lipid II and which is produced by Bacillus sp. strain HIL Y-85,54728. The structural gene of mersacidin as well as accessory genes is organized in a biosynthetic gene cluster which is located on the chromosome and contains three open reading frames with similarities to regulatory proteins: mrsR2 and mrsK2 encode two proteins with homology to bacterial two-component systems, and mrsR1 shows similarity to a response regulator. Both mrsR2/K2 and mrsR1 were inactivated by insertion of an antibiotic resistance marker. Disruption of mrsR1 resulted in loss of mersacidin production; however, producer self-protection was not impaired. In contrast, inactivation of mrsR2/K2 led to an increased susceptibility to mersacidin whereas biosynthesis of the lantibiotic remained unaffected. Binding of mersacidin to intact cells was significantly enhanced in the mrsR2/K2 knockout mutant. Reverse transcription-PCR analysis from total RNA preparations showed that in contrast to the wild-type strain, the structural genes of the ABC transporter MrsFGE were not transcribed in the knockout mutant. It was therefore concluded that producer self-protection against mersacidin is conferred by the ABC transporter MrsFGE and that the transcription of mrsFGE is regulated by MrsR2/K2, whereas production of the antibacterial peptide is solely activated by MrsR1.

Site-directed mutagenesis of hGH at the 54–74 loop selectively modifies its lactogenic receptor-mediated biological activity

1993 ◽  
Vol 97 (1-2) ◽  
pp. 129-134 ◽  
Author(s):  
Edna Sakal ◽  
Amir Tchelet ◽  
Eriko Uchida ◽  
Seitaro Shimokawa ◽  
Satoshi Nishikawa ◽  
...  
Keyword(s):  
Biological Activity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

scholarly journals Site-directed mutagenesis of monocyte chemoattractant protein-1 identifies two regions of the polypeptide essential for biological activity

1996 ◽  
Vol 313 (2) ◽  
pp. 633-640 ◽  
Author(s):  
Clifford J. BEALL ◽  
Sangeeta MAHAJAN ◽  
Donald E. KUHN ◽  
Pappachan E. KOLATTUKUDY
Keyword(s):  
Biological Activity ◽  
Inflammatory Diseases ◽  
Expression System ◽  
Point Mutations ◽  
Site Directed Mutagenesis ◽  
Monocyte Chemoattractant Protein 1 ◽  
Monocyte Chemoattractant ◽  
Monocyte Chemoattractant Protein ◽  
Monocyte Migration ◽  
Important Region

Monocyte chemoattractant protein-1 (MCP-1) mediates monocyte migration into tissues in inflammatory diseases and atherosclerosis. We have investigated structure-activity relationships for human MCP-1. Mutations were introduced based upon differences between MCP-1 and the structurally related but functionally distinct molecule interleukin-8 (IL-8). Mutant proteins produced using the baculovirus/insect cell expression system were purified and their ability to stimulate monocyte chemotaxis and elevation of intracellular calcium in THP-1 monocytic leukaemia cells was measured. Two regions in MCP-1 were identified as important for its biological activity. One region consists of the sequence Thr-Cys-Cys-Tyr (amino acids 10-13). Point mutations of Thr-10 to Arg and Tyr-13 to Ile greatly lowered MCP-1 activity. The second functionally important region is formed by Ser-34 and Lys-35. Insertion of a Pro between these two residues, or their substitution by the sequence Gly-Pro-His, caused nearly complete loss of MCP-1 activity. Competition binding experiments showed that the mutations that affected activity also lowered the ability to compete with wild-type MCP-1 for receptors on THP-1 cells. Point mutations at positions 8, 15, 30, 37, 38 and 68 had little effect on MCP-1 activity. The important regions that we have identified in MCP-1 correspond with previously identified functionally important regions of IL-8, suggesting that the two molecules bind to their respective receptors by similar contacts.

scholarly journals Site-directed mutagenesis of the leech-derived factor Xa inhibitor antistasin. Probing of the reactive site

1992 ◽  
Vol 287 (3) ◽  
pp. 943-949 ◽  
Author(s):  
K J Hofmann ◽  
E M Nutt ◽  
C T Dunwiddie
Keyword(s):  
Amino Acid ◽  
Proteinase Inhibitors ◽  
Expression System ◽  
Factor Xa ◽  
Site Directed Mutagenesis ◽  
Arginine Residue ◽  
Common Mechanism ◽  
Directed Mutagenesis ◽  
Reactive Site ◽  
Terminal Domain

Antistasin (ATS) is a leech-derived 119-amino-acid protein which exhibits potent and highly selective inhibition of coagulation Factor Xa. It inhibits Factor Xa according to a common mechanism of serine-proteinase inhibitors in which a conformationally rigid substrate-like reactive site is presented to the enzyme. In this study a recombinant version of ATS was expressed and purified utilizing a yeast expression system in order to probe the reactive site P1 (Arg-34) and P1′ (Val-35) residues by site-directed mutagenesis. The results demonstrate the requirement for a positively charged residue in the P1 position of ATS, with an arginine residue preferred over a lysine, yielding K1 values of 61 pM and 1.28 nM respectively. Mutation of the P1 arginine residue to the non-polar amino acid leucine abolished its inhibitory potency toward Factor Xa. The role of the C-terminal domain of ATS, which shares significant amino acid sequence identity with the N-terminal domain, was investigated by creating a second reactive site in the corresponding position of the C-terminal domain. The inhibitory activity of this mutant demonstrated that the C-terminal domain of ATS is not folded into the proper conformation necessary to create a functional inhibitory domain.

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