scholarly journals Improvement of Posttranslational Bottlenecks in the Production of Penicillin Amidase in Recombinant Escherichiacoli Strains

2003 ◽  
Vol 69 (2) ◽  
pp. 1237-1245 ◽  
Author(s):  
Z. Ignatova ◽  
A. Mahsunah ◽  
M. Georgieva ◽  
V. Kasche
Keyword(s):  
Bacterial Culture ◽  
Signal Sequence ◽  
Peptide Sequence ◽  
Extracellular Medium ◽  
Penicillin Amidase ◽  
Signal Peptide Sequence ◽  
Threefold Increase ◽  
Translocation Efficiency ◽  
Protease Deficient ◽  
Selection Of

ABSTRACT Using periplasmic penicillin amidase (PA) from Escherichia coli ATCC 11105 as a model recombinant protein, we reviewed the posttranslational bottlenecks in its overexpression and undertook attempts to enhance its production in different recombinant E. coli expression hosts. Intracellular proteolytic degradation of the newly synthesized PA precursor and translocation through the plasma membrane were determined to be the main posttranslational processes limiting enzyme production. Rate constants for both intracellular proteolytic breakdown (kd ) and transport (kt ) were used as quantitative tools for selection of the appropriate host system and cultivation medium. The production of mature active PA was increased up to 10-fold when the protease-deficient strain E. coli BL21(DE3) was cultivated in medium without a proteinaceous substrate, as confirmed by a decrease in the sum of the constants kd and kt . The original signal sequence of pre-pro-PA was exchanged with the OmpT signal peptide sequence in order to increase translocation efficiency; the effects of this change varied in the different E. coli host strains. Furthermore, we established that simultaneous coexpression of the OmpT pac gene with some proteins of the Sec export machinery of the cell resulted in up to threefold-enhanced PA production. In parallel, we made efforts to increase PA flux via coexpression with the kil gene (killing protein). The primary effects of the kil gene were the release of PA into the extracellular medium and an approximately threefold increase in the total amount of PA produced per liter of bacterial culture.

scholarly journals Association of the leucine-7 to proline-7 variation in the signal sequence of neuropeptide Y with major depression

2012 ◽  
Vol 24 (2) ◽  
pp. 81-90 ◽  
Author(s):  
Pernille Koefoed ◽  
David P.D. Woldbye ◽  
Thomas v. O. Hansen ◽  
Lene F. Eplov ◽  
Søren H. Christiansen ◽  
...  
Keyword(s):  
Major Depression ◽  
Neuropeptide Y ◽  
Signal Sequence ◽  
Mrna Level ◽  
Peptide Sequence ◽  
Psychiatric Disease ◽  
Danish Population ◽  
Signal Peptide Sequence ◽  
Functional Consequences

Objective:There is clear evidence of a genetic component in major depression, and several studies indicate that neuropeptide Y (NPY) could play an important role in the pathophysiology of the disease. A well-known polymorphism encoding the substitution of leucine to proline in the signal peptide sequence of NPY (Leu7Pro variation) was previously found to protect against depression. Our study aimed at replicating this association in a large Danish population with major depression.Method:Leu7Pro was studied in a sample of depressed patients and ethnically matched controls, as well as psychiatric disease controls with schizophrenia. Possible functional consequences of Leu7Pro were exploredin vitro.Results:In contrast to previous studies, Pro7 appeared to be a risk allele for depression, being significantly more frequent in the depression sample (5.5%,n= 593;p= 0.009; odds ratio, OR: 1.46) as compared to ethnically matched controls (3.8%,n= 2912), while schizophrenia patients (4.1%,n= 503) did not differ.In vitro, the Pro7 substitution appeared to be associated with reduced levels of NPY without affecting its mRNA level.Conclusion:The Leu7Pro variation may increase the risk of major depression, possibly by affecting the biosynthesis of NPY.

scholarly journals Role of Signal Sequence in Vaccine-Induced Protection against Experimental Coccidioidomycosis

2002 ◽  
Vol 70 (7) ◽  
pp. 3539-3545 ◽  
Author(s):  
Chengyong Jiang ◽  
D. Mitchell Magee ◽  
F. Douglas Ivey ◽  
Rebecca A. Cox
Keyword(s):  
Signal Peptide ◽  
Synthetic Peptide ◽  
Gene Sequence ◽  
Signal Sequence ◽  
Lethal Dose ◽  
Full Length ◽  
Peptide Sequence ◽  
Coccidioides Immitis ◽  
Signal Peptide Sequence ◽  
Gene Vaccine

ABSTRACT The vaccine efficacy of the gene sequence encoding the signal peptide of the antigen known as antigen 2 or proline-rich antigen (Ag2/PRA), an immunodominant antigen present in the cell wall of the fungal pathogen Coccidioides immitis, was investigated in a murine model of coccidioidomycosis. Expression plasmids for Ag2/PRA(1-18) DNA (signal sequence), Ag2/PRA(19-194) DNA (lacking the signal sequence), and Ag2/PRA(1-194) DNA (full length) were inserted in the pVR1012 vector, and the constructs were used to vaccinate the highly susceptible BALB/c mouse strain. Immunization with the signal gene sequence significantly reduced the fungal burden in the lungs and spleens of mice 12 days after intraperitoneal challenge with a lethal dose of 2,500 C. immitis arthroconidia, to a level comparable to the protection induced in mice immunized with the full-length Ag2/PRA(1-194) DNA. The Ag2/PRA(19-194) gene protected mice but to a significantly lower level than the signal sequence or the full-length Ag2 gene. The immunizing capacity of Ag2/PRA(1-18) was not attributable to a nonspecific immunostimulatory effect of DNA, as evidenced by the fact that mice immunized with a frameshift mutation of Ag2/PRA(1-18) were not protected against challenge. Furthermore, a synthetic peptide corresponding to the translated sequence of Ag2/PRA(1-18) DNA protected mice, albeit at a lower level than the Ag2/PRA(1-18) DNA vaccine. The protection induced with the signal gene vaccine correlated with the production of gamma interferon when splenocytes from Ag2/PRA(1-18)-immunized mice were stimulated with recombinant full-length Ag2 and was not associated with the production of anti-Coccidioides immunoglobulin G antibody. This is the first study to establish that a signal peptide sequence alone, administered as a gene vaccine or synthetic peptide, can induce protective immunity against a microbial pathogen.

scholarly journals Characterization of a Unique Group-Specific Protein (U122) of the Severe Acute Respiratory Syndrome Coronavirus

2004 ◽  
Vol 78 (14) ◽  
pp. 7311-7318 ◽  
Author(s):  
Burtram C. Fielding ◽  
Yee-Joo Tan ◽  
Shen Shuo ◽  
Timothy H. P. Tan ◽  
Eng-Eong Ooi ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Signal Sequence ◽  
Transmembrane Helix ◽  
Specific Protein ◽  
Open Reading Frames ◽  
Peptide Sequence ◽  
Type I ◽  
Signal Peptide Sequence ◽  
Acid Protein ◽  
Novel Coronavirus

ABSTRACT A novel coronavirus (CoV) has been identified as the etiological agent of severe acute respiratory syndrome (SARS). The SARS-CoV genome encodes the characteristic essential CoV replication and structural proteins. Additionally, the genome contains six group-specific open reading frames (ORFs) larger than 50 amino acids, with no known homologues. As with the group-specific genes of the other CoVs, little is known about the SARS-CoV group-specific genes. SARS-CoV ORF7a encodes a putative unique 122-amino-acid protein, designated U122 in this study. The deduced sequence contains a probable cleaved signal sequence and a C-terminal transmembrane helix, indicating that U122 is likely to be a type I membrane protein. The C-terminal tail also contains a typical endoplasmic reticulum (ER) retrieval motif, KRKTE. U122 was expressed in SARS-CoV-infected Vero E6 cells, as it could be detected by Western blot and immunofluorescence analyses. U122 is localized to the perinuclear region of both SARS-CoV-infected and transfected cells and colocalized with ER and intermediate compartment markers. Mutational analyses showed that both the signal peptide sequence and ER retrieval motif were functional.

scholarly journals An Experimental Approach for the Identification of Conserved Secreted Proteins in Trypanosomatids

2010 ◽  
Vol 2010 ◽  
pp. 1-13 ◽  
Author(s):  
Rosa M. Corrales ◽  
Françoise Mathieu-Daudé ◽  
Déborah Garcia ◽  
Simone F. Brenière ◽  
Denis Sereno
Keyword(s):  
Secretory Pathway ◽  
Experimental System ◽  
Bioinformatic Analysis ◽  
Extracellular Proteins ◽  
Peptide Sequence ◽  
Secreted Proteins ◽  
Extracellular Medium ◽  
Signal Peptide Sequence ◽  
A Genome ◽  
Genes Encoding

Extracellular factors produced byLeishmania spp., Trypanosoma cruzi,andTrypanosoma bruceiare important in the host-parasite relationship. Here, we describe a genome-based approach to identify putative extracellular proteins conserved among trypanosomatids that are likely involved in the classical secretory pathway. Potentially secreted proteins were identified by bioinformatic analysis of theT. cruzigenome. A subset of thirteen genes encoding unknown proteins with orthologs containing a signal peptide sequence inL. infantum, L. major,andT. bruceiwere transfected intoL. infantum. Tagged proteins detected in the extracellular medium confirmed computer predictions in about 25% of the hits. Secretion was confirmed for twoL. infantumorthologs proteins using the same experimental system. Infectivity studies of transgenicLeishmaniaparasites suggest that one of the secreted proteins increases parasite replication inside macrophages. This methodology can identify conserved secreted proteins involved in the classical secretory pathway, and they may represent potential virulence factors in trypanosomatids.

Molecular cloning of a 47 kDa tissue-specific and differentiation-dependent urothelial cell surface glycoprotein

1993 ◽  
Vol 106 (1) ◽  
pp. 31-43 ◽  
Author(s):  
X.R. Wu ◽  
T.T. Sun
Keyword(s):  
Transmembrane Domain ◽  
Core Protein ◽  
Surface Glycoprotein ◽  
Peptide Sequence ◽  
Type I ◽  
Apical Surface ◽  
Bladder Epithelium ◽  
Cell Surface Glycoprotein ◽  
Signal Peptide Sequence ◽  
C Terminus

Despite the fact that bladder epithelium has many interesting biological features and is a frequent site of carcinoma formation, relatively little is known about its biochemical differentiation. We have shown recently that a 47 kDa glycoprotein, uroplakin III (UPIII), in conjunction with uroplakins I (27 kDa) and II (15 kDa), forms the asymmetric unit membrane (AUM)--a highly specialized biomembrane characteristic of the apical surface of bladder epithelium. Deglycosylation and cDNA sequencing revealed that UPIII contains up to 20 kDa of N-linked sugars attached to a core protein of 28.9 kDa. The presence of an N-terminal signal peptide sequence and a single transmembrane domain located near the C terminus, plus the N-terminal location of all the potential N-glycosylation sites, points to a type I (N-exo/C-cyto) configuration. Thus the mass of the extracellular domain (20 kDa plus up to 20 kDa of sugar) of UPIII greatly exceeds that of its intracellular domain (5 kDa). Such an asymmetrical mass distribution, a feature shared by the other two major uroplakins, provides a molecular explanation as to why the luminal leaflet of AUM is almost twice as thick as the cytoplasmic one. The fact that of the three major proteins of AUM only UPIII has a significant cytoplasmic domain suggests that this molecule may play an important role in AUM-cytoskeleton interaction in terminally differentiated urothelial cells.

scholarly journals Amino acid sequence of the penicillin-binding protein/dd-peptidase of Streptomyces K15. Predicted secondary structures of the low Mr penicillin-binding proteins of class A

1991 ◽  
Vol 279 (1) ◽  
pp. 223-230 ◽  
Author(s):  
P Palomeque-Messia ◽  
S Englebert ◽  
M Leyh-Bouille ◽  
M Nguyen-Distèche ◽  
C Duez ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Residue ◽  
Active Sites ◽  
Binding Protein ◽  
Secondary Structures ◽  
Peptide Sequence ◽  
Penicillin Binding Protein ◽  
Signal Peptide Sequence ◽  
Beta Lactamases ◽  
Class A

The low-Mr penicillin-binding protein (PBP)/DD-transpeptidase of Streptomyces K15 is synthesized in the form of a 291-amino acid-residue precursor possessing a cleavable 29-amino acid-residue signal peptide. Sequence-similarity searches and hydrophobic-cluster analysis show that the Streptomyces K15 enzyme, the Escherichia coli PBPs/DD-carboxy-peptidases 5 and 6, the Bacillus subtilis PBP/DD-carboxypeptidase 5 and the spoIIA product (a putative PBP involved in the sporulation of B. subtilis) are structurally related and form a distinct class A of low-Mr PBPs/DD-peptidases. The distribution of the hydrophobic clusters along the amino acid sequences also shows that the Streptomyces K15 PBP, and by extension the other PBPs of class A, have similarity in the polypeptide folding, with the beta-lactamases of class A, with as reference the Streptomyces albus G and Staphylococcus aureus beta-lactamases of known three-dimensional structure. This comparison allows one to predict most of the secondary structures in the PBPs and the amino acid motifs that define the enzyme active sites.

Cassette mutagenic analysis of the yeast invertase signal peptide: effects on protein translocation

1989 ◽  
Vol 9 (8) ◽  
pp. 3400-3410
Author(s):  
J K Ngsee ◽  
W Hansen ◽  
P Walter ◽  
M Smith
Keyword(s):  
Signal Peptide ◽  
Protein Translocation ◽  
Plasmid Vector ◽  
Peptide Sequence ◽  
Secretory Process ◽  
Hydrophobic Core ◽  
Class Iii ◽  
Yeast Plasmid ◽  
Signal Peptide Sequence ◽  
Transcription Terminator

The coding sequence of the SUC2 locus was placed under the control of the constitutive ADH1 promoter and transcription terminator in a centromere-based yeast plasmid vector from which invertase is expressed in a Suc- strain of Saccharomyces cerevisiae. Mutants in the signal peptide sequence were produced by replacing this region of the gene with synthetic oligonucleotide cassettes containing mixtures of nucleotides at several positions. The mutants could be divided into three classes on the basis of the ability to secrete invertase. Class I mutants produced secreted invertase but in reduced amount. The class II mutant, 4-55B, also exhibited reduced a level of invertase, but a significant fraction of the enzyme was intracellular. Class III mutants were partially defective in translocation from the cytoplasm to the endoplasmic reticulum and produced enzymatically active, unglycosylated preinvertase in the cytoplasm. Class III mutant preinvertases were also defective in translocation across canine pancreas microsomes. These results suggested that the reduced level of invertase resulted from proteolytic degradation of inefficiently transported intermediates. Comparison of the sequences of the mutant signal peptides indicated that amino acids at the extreme amino terminus and adjacent to the cleavage site play a crucial role in the secretory process when combined with a mutation within the hydrophobic core.

scholarly journals Cell-free synthesis of the hirudin variant 1 of the blood-sucking leech Hirudo medicinalis

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Doreen A. Wüstenhagen ◽  
Phil Lukas ◽  
Christian Müller ◽  
Simone A. Aubele ◽  
Jan-Peter Hildebrandt ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Peptide Sequence ◽  
Bacterial Cells ◽  
Recombinant Hirudin ◽  
Signal Peptide Sequence ◽  
Cell Lysates ◽  
Blood Sucking ◽  
A Cell ◽  
Synthesis Approach ◽  
Cell Free Protein Synthesis

AbstractSynthesis and purification of peptide drugs for medical applications is a challenging task. The leech-derived factor hirudin is in clinical use as an alternative to heparin in anticoagulatory therapies. So far, recombinant hirudin is mainly produced in bacterial or yeast expression systems. We describe the successful development and application of an alternative protocol for the synthesis of active hirudin based on a cell-free protein synthesis approach. Three different cell lysates were compared, and the effects of two different signal peptide sequences on the synthesis of mature hirudin were determined. The combination of K562 cell lysates and the endogenous wild-type signal peptide sequence was most effective. Cell-free synthesized hirudin showed a considerably higher anti-thrombin activity compared to recombinant hirudin produced in bacterial cells.

Molecular cloning and sequence analysis of putative chicken prolactin cDNA

1989 ◽  
Vol 2 (1) ◽  
pp. 21-30 ◽  
Author(s):  
M.C. Hanks ◽  
J.A. Alonzi ◽  
P.J. Sharp ◽  
H.M. Sang
Keyword(s):  
Amino Acids ◽  
Genomic Dna ◽  
Signal Sequence ◽  
Peptide Sequence ◽  
Mrna Levels ◽  
Reading Frame ◽  
Ring Dove ◽  
Prolactin Gene ◽  
Pituitary Glands ◽  
High Degree

ABSTRACT A cDNA library was prepared from mRNA isolated from anterior pituitary glands of incubating bantam hens, in which prolactin mRNA levels were predicted to be very high. Nine clones, representing abundant mRNA species, were identified and shown to contain homologous sequences. Two clones, of 871 bp and 580 bp, were analysed by DNA sequencing. The shorter clone was found to be a truncated cDNA product but otherwise identical to the longer clone. The 871 bp cDNA, PRL101, contains an open reading frame capable of encoding a polypeptide of 229 amino acids. This putative polypeptide has a high degree of homology to mammalian prolactins (approximately 70%), strongly suggesting that PRL101 encodes chicken preprolactin. The protein was predicted to have a 30 amino acid signal sequence which would be cleaved off to give a mature protein of 199 amino acids. The peptide sequence also had a 26% homology to chicken growth hormone, which is related to prolactin. This similarity confirms the conclusion that PRL101 is a chicken prolactin cDNA clone. An abundant mRNA of approximately 880 b was detected in poly(A)+ RNA from pituitary glands probed with PRL101. Analysis of chicken genomic DNA showed that there is one copy of the prolactin gene in the genome. PRL101 hybridized strongly to genomic DNA from closely related galliforms (quail and turkey) and less strongly to DNA from more distantly related species (duck and ring dove).

Thaumatin Production in Aspergillus awamori by Use of Expression Cassettes with Strong Fungal Promoters and High Gene Dosage

1999 ◽  
Vol 65 (3) ◽  
pp. 1168-1174 ◽  
Author(s):  
Francisco-Jose Moralejo ◽  
Rosa-Elena Cardoza ◽  
Santiago Gutierrez ◽  
Juan F. Martin
Keyword(s):  
Protein Translocation ◽  
Gene Dosage ◽  
Signal Sequence ◽  
Aspergillus Awamori ◽  
Recognition Sequence ◽  
Transcript Levels ◽  
Sweet Protein ◽  
High Gene ◽  
Protease Deficient ◽  
Fungal Promoters

ABSTRACT Four expression cassettes containing strong fungal promoters, a signal sequence for protein translocation, a KEX protease cleavage site, and a synthetic gene (tha) encoding the sweet protein thaumatin II were used to overexpress this protein in Aspergillus awamori lpr66, a PepA protease-deficient strain. The best expression results were obtained with the gdhA promoter ofA. awamori or with the gpdA promoter ofAspergillus nidulans. There was good correlation oftha gene dosage, transcript levels, and thaumatin secretion. The thaumatin gene was expressed as a transcript of the expected size in each construction (1.9 or 1.4 kb), and the transcript levels and thaumatin production rate decayed at the end of the growth phase, except in the double transformant TB2b1-44-GD5, in which secretion of thaumatin continued until 96 h. The recombinant thaumatin secreted by a high-production transformant was purified to homogeneity, giving one major component and two minor components. In all cases, cleavage of the fused protein occurred at the KEX recognition sequence. This work provides new expression systems inA. awamori that result in very high levels of thaumatin production.

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