Functional expression of the yeast Mn-superoxide dismutase gene in Escherichia coli requires deletion of the signal peptide sequence

Gene ◽  
1988 ◽  
Vol 73 (1) ◽  
pp. 121-130 ◽  
Author(s):  
Irene S. Schrank ◽  
Paul F.G. Sims ◽  
Stephen G. Oliver
Keyword(s):  
Escherichia Coli ◽  
Superoxide Dismutase ◽  
Signal Peptide ◽  
Functional Expression ◽  
Peptide Sequence ◽  
Signal Peptide Sequence ◽  
Superoxide Dismutase Gene

scholarly journals Revised Translation Start Site for secM Defines an Atypical Signal Peptide That Regulates Escherichia coli secA Expression

2000 ◽  
Vol 182 (19) ◽  
pp. 5592-5595 ◽  
Author(s):  
Shameema Sarker ◽  
Kenneth E. Rudd ◽  
Donald Oliver
Keyword(s):  
Escherichia Coli ◽  
Signal Peptide ◽  
Peptide Sequence ◽  
Start Site ◽  
Signal Peptide Sequence ◽  
Amino Terminal ◽  
Upstream Regulator ◽  
Translation Start ◽  
Responsive Regulation ◽  
Translational Start

ABSTRACT The secretion-responsive regulation of Escherichia coli secA occurs by coupling its translation to the translation and secretion of an upstream regulator, secM (formerly geneX). We revise the translational start site for secM, defining a new signal peptide sequence with an extended amino-terminal region. Mutational studies indicate that certain atypical amino acyl residues within this extended region are critical for propersecA regulation.

In Silico Investigation of Signal Peptide Sequences to Enhance Secretion of CD44 Nanobodies Expressed in Escherichia coli

2020 ◽  
Vol 21 ◽  
Author(s):  
Soudabeh Kavousipour ◽  
Shiva Mohammadi ◽  
Ebrahim Eftekhar ◽  
Mahdi Barazesh ◽  
Mohammad Hossein Morowvat
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Signal Peptide ◽  
In Silico ◽  
Extracellular Space ◽  
Peptide Sequence ◽  
Signal Peptides ◽  
Signal Peptide Sequence ◽  
E Coli ◽  
Peptide Database

Background: The selection of a suitable signal peptide that can direct recombinant proteins from the cytoplasm to the extracellular space is an important criterion affecting the production of recombinant proteins in Escherichia coli, a widely used host. Nanobodies are currently attracting the attention of scientists as antibody alternatives due to their specific properties and feasibility of production in E. coli. Objective: CD44 nanobodies constitute a potent therapeutic agent that can block CD44/HA interaction in cancer and inflammatory diseases. This molecule may also function as a drug against cancer cells and has been produced previously in E. coli without a signal peptide sequence. The goal of this project was to find a suitable signal peptide to direct CD44 nanobody extracellular secretion in E. coli that will potentially lead to optimization of experimental methods and facilitate downstream steps such as purification. Methods: We analyzed 40 E. coli derived signal peptides retrieved from the Signal Peptide database and selected the best candidate signal peptides according to relevant criteria including signal peptide probability, stability, and physicochemical features, which were evaluated using signalP software version 4.1 and the ProtParam tool, respectively. Results: In this in silico study, suitable candidate signal peptide(s) for CD44 nanobody secretory expression were identified. CSGA, TRBC, YTFQ, NIKA, and DGAL were selected as appropriate signal peptides with acceptable D-scores, and appropriate physicochemical and structural properties. Following further analysis, TRBC was selected as the best signal peptide to direct CD44 nanobody expression to the extracellular space of E. coli. Conclusion: The selected signal peptide, TRBC is the most suitable to promote high level secretory production of CD44 nanobodies in E. coli and potentially will be useful for scaling up CD44 nanobody production in experimental research as well as in other CD44 nanobody applications. However, experimental work is needed to confirm the data.

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.

Production of 12-hydroxy dodecanoic acid methyl ester using a signal peptide sequence-optimized transporter AlkL and a novel monooxygenase

2019 ◽  
Vol 291 ◽  
pp. 121812 ◽  
Author(s):  
Hee-Wang Yoo ◽  
Joonwon Kim ◽  
Mahesh D. Patil ◽  
Beom Gi Park ◽  
Sung-yeon Joo ◽  
...  
Keyword(s):  
Methyl Ester ◽  
Signal Peptide ◽  
Acid Methyl Ester ◽  
Peptide Sequence ◽  
Dodecanoic Acid ◽  
Signal Peptide Sequence ◽  
Acid Methyl

scholarly journals A New IL6 Isoform in Chinese Soft-Shelled Turtle (Pelodiscus sinesis) Discovered: Its Regulation during Cold Stress and Infection

Biology ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 111
Author(s):  
Zuobing Zhang ◽  
Miao Tian ◽  
Ruxin Song ◽  
Xiao Xing ◽  
Yong Fan ◽  
...  
Keyword(s):  
Cold Stress ◽  
Signal Peptide ◽  
Genomic Sequence ◽  
Adaptive Immune Response ◽  
Environmental Water ◽  
Peptide Sequence ◽  
Signal Peptide Sequence ◽  
Gene Characterization ◽  
Peptide Gene ◽  
Response To Temperature

The Chinese soft-shelled turtle (Pelodiscus sinesis) is a widely cultured commercial species in East and Southeast Asian countries. The turtles frequently suffer from acute cold stress during farming in China. Stress-induced factor such as Interleukin-6 (IL6) is a multifunctional molecule that plays important roles in innate and adaptive immune response. In the present study, we found that the turtle possessed two IL6 transcripts, where one IL6 transcript contained a signal peptide sequence (psIL6), while the other IL6 transcript (psIL6ns) possessed no such signal peptide gene. To test any differential expression of the two isoforms during temperature and microbial stress, turtles were adapted to optimal environmental water temperature (25 °C), stressed by acute cooling for 24 h, followed with the challenge of Aeromonas hydrophila (1.8 × 108 CFU) or Staphylococcus aureus (5.8 × 108 CFU). Gene characterization revealed that psIL6ns, a splicer without codons encoding a signal peptide and identical to the one predicted from genomic sequence, and psIL6, a splicer with codons encoding a signal peptide, were both present. Inducible expression was documented in primary spleen cells stimulated with ConA and poly I: C. The splenic and intestinal expression of psIL6ns and psIL6 was increased in response to temperature stress and bacterial infection.

scholarly journals Characterization of PauB, a Novel Broad-Spectrum Plasminogen Activator from Streptococcus uberis

2002 ◽  
Vol 184 (1) ◽  
pp. 119-125 ◽  
Author(s):  
Philip N. Ward ◽  
James A. Leigh
Keyword(s):  
Plasminogen Activator ◽  
Signal Peptide ◽  
Open Reading Frame ◽  
Peptide Sequence ◽  
Expression Cloning ◽  
Signal Peptide Sequence ◽  
Putative Gene ◽  
Reading Frame ◽  
Streptococcus Uberis ◽  
Broad Specificity

ABSTRACT A bovine plasminogen activator of atypical molecular mass (∼45 kDa) from Streptococcus uberis strain SK880 had been identified previously (L. B. Johnsen, K. Poulsen, M. Kilian, and T. E. Petersen. Infect. Immun. 67:1072–1078, 1999). The strain was isolated from a clinical case of bovine mastitis. The isolate was found not to secrete PauA, a bovine plasminogen activator expressed by the majority of S. uberis strains. Analysis of the locus normally occupied by pauA revealed an absence of the pauA open reading frame. However, an alternative open reading frame was identified within the same locus. Sequence analysis of the putative gene suggested limited but significant homology to other plasminogen activators. A candidate signal peptide sequence and cleavage site were also identified. Expression cloning of DNA encoding the predicted mature protein (lacking signal peptide) confirmed that the open reading frame encoded a plasminogen activator of the expected size, which we have named PauB. Both native and recombinant forms of PauB displayed an unexpectedly broad specificity profile for bovine, ovine, equine, caprine, porcine, rabbit, and human plasminogen. Clinical and nonclinical field isolates from nine United Kingdom sites were screened for the pauB gene and none were identified as carrying it. Similarly, clinical isolates from 20 Danish herds were all found to encode PauA and not PauB. Therefore, PauB represents a novel but rare bacterial plasminogen activator which displays very broad specificity.

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