scholarly journals A Combination Strategy of Solubility Enhancers for Effective Production of Soluble and Bioactive Human Enterokinase

2021 ◽  
Author(s):  
Jinhak Kwon ◽  
Hyeongjun Cho ◽  
Seungmin Kim ◽  
Yiseul Ryu ◽  
Joong-jae Lee
Keyword(s):  
Recombinant Proteins ◽  
Phosphoglycerate Kinase ◽  
Combinatorial Design ◽  
Peptide Sequence ◽  
Protein Digestion ◽  
Combination Strategy ◽  
Enhanced Solubility ◽  
Production Strategy ◽  
Solubility Enhancers ◽  
Specific Peptide

Enterokinase is one of the hydrolases that catalyze hydrolysis to regulate biological processes in intestinal visceral mucosa. Enterokinase plays an essential role in accelerating the process of protein digestion as it converts trypsinogen into active trypsin by accurately recognizing and cleaving a specific peptide sequence, (Asp)4-Lys. Due to its exceptional substrate specificity, enterokinase is widely used as a versatile molecular tool in various bioprocessing, especially in removing fusion tags from recombinant proteins. Despite its biotechnological importance, mass production of soluble enterokinase in bacteria still remains an unsolved challenge. Here, we present an effective production strategy of human enterokinase using tandemly linked solubility enhancers consisting of thioredoxin, phosphoglycerate kinase or maltose-binding protein. The resulting enterokinases exhibited significantly enhanced solubility and bacterial expression level while retaining enzymatic activity, which demonstrates that combinatorial design of fusion proteins has the potential to provide an efficient way to produce recombinant proteins in bacteria.

scholarly journals The Chlamydomonas Dhc1 gene encodes a dynein heavy chain subunit required for assembly of the I1 inner arm complex.

1997 ◽  
Vol 8 (4) ◽  
pp. 607-620 ◽  
Author(s):  
S H Myster ◽  
J A Knott ◽  
E O'Toole ◽  
M E Porter
Keyword(s):  
Heavy Chain ◽  
Insertional Mutagenesis ◽  
Northern Blot Analysis ◽  
Immunoblot Analysis ◽  
Transcription Unit ◽  
Peptide Sequence ◽  
Dynein Heavy Chain ◽  
The Individual ◽  
Intermediate Chains ◽  
Specific Peptide

Multiple members of the dynein heavy chain (Dhc) gene family have been recovered in several organisms, but the relationships between these sequences and the Dhc isoforms that they encode are largely unknown. To identify Dhc loci and determine the specific functions of the individual Dhc isoforms, we have screened a collection of motility mutants generated by insertional mutagenesis in Chlamydomonas. In this report, we characterize one strain, pf9-3, in which the insertion event was accompanied by a deletion of approximately 13 kb of genomic DNA within the transcription unit of the Dhc1 gene. Northern blot analysis confirms that pf9-3 is a null mutation. Biochemical and structural studies of isolated axonemes demonstrate that the pf9-3 mutant fails to assemble the I1 inner arm complex, a two-headed dynein isoform composed of two Dhcs (1 alpha and 1 beta) and three intermediate chains. To determine if the Dhc1 gene product corresponds to one of the Dhcs of the I1 complex, antibodies were generated against a Dhc1-specific peptide sequence. Immunoblot analysis reveals that the Dhc1 gene encodes the 1 alpha Dhc subunit. These studies thus, identify the first inner arm Dhc locus to be described in any organism and further demonstrate that the 1 alpha Dhc subunit plays an essential role in the assembly of the I1 inner arm complex.

Metacognition of Problem-Solving Strategies in Brazil, India, and the United States

2007 ◽  
Vol 7 (1-2) ◽  
pp. 1-25 ◽  
Author(s):  
Brian Wiley ◽  
C. Dominik Güss
Keyword(s):  
United States ◽  
Problem Solving ◽  
The United States ◽  
Uncertainty Avoidance ◽  
Metacognitive Strategies ◽  
Thought Processes ◽  
Combination Strategy ◽  
Problem Solving Strategies ◽  
Production Strategy ◽  
The U.S

AbstractMetacognition, the observation of one's own thinking, is a key cognitive ability that allows humans to influence and restructure their own thought processes. The influence of culture on metacognitive strategies is a relatively new topic. Using Antonietti's, Ignazi's and Perego's questionnaire on metacognitive knowledge about problem-solving strategies (2000), five strategies in three life domains were assessed among student samples in Brazil, India, and the United States (N=317), regarding the frequency, facility, and efficacy of these strategies. To investigate cross-cultural similarities and differences in strategy use, nationality and uncertainty avoidance values were independent variables. Uncertainty avoidance was expected to lead to high frequency of decision strategies. However, results showed no effect of uncertainty avoidance on frequency, but an effect on facility of metacognitive strategies. Comparing the three cultural samples, all rated analogy as the most frequent strategy. Only in the U.S. sample, analogy was also rated as the most effective and easy to apply strategy. Every cultural group showed a different preference regarding what metacognitive strategy was most effective. Indian participants found the free production strategy to be more effective, and Indian and Brazilian participants found the combination strategy to be more effective compared to the U.S. participants. As key abilities for the five strategies, Indians rated speed, Brazilians rated synthesis, and U.S. participants rated critical thinking as more important than the other participants. These results reflect the embedded nature and functionality of problem solving strategies in specific cultural environments. The findings will be discussed referring to an eco-cultural framework.

scholarly journals Infrequent Occurrence of Natural Mutations in the pp65495–503 Epitope Sequence Presented by the HLA A∗0201 Allele among Human Cytomegalovirus Isolates

2001 ◽  
Vol 75 (5) ◽  
pp. 2472-2474 ◽  
Author(s):  
John A. Zaia ◽  
Ghislaine Gallez-Hawkins ◽  
Xiuli Li ◽  
Zhi-Qiang Yao ◽  
Norma Lomeli ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Peptide Sequence ◽  
Amino Terminus ◽  
Methionine Residue ◽  
Rare Mutations ◽  
Epitope Sequence ◽  
Human Cmv ◽  
Specific Peptide

ABSTRACT To determine if mutations of an immunodominant HLA-restricted cytomegalovirus (CMV) peptide sequence occur in nature, the sequence corresponding to the HLA A∗0201-specific peptide CMVpp65495–503 was determined in 50 human CMV isolates. Rare mutations were detected; 6 of 50 were silent mutations at the amino terminus of the peptide, while 3 of 50 were mutations of the native methionine residue to isoleucine (M499I). The observed M499I mutation in three isolates decreased cytolytic targeting.

scholarly journals A predictive model for vertebrate bone identification from collagen using proteomic mass spectrometry

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Heyi Yang ◽  
Erin R. Butler ◽  
Samantha A. Monier ◽  
Jennifer Teubl ◽  
David Fenyö ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Species Identification ◽  
Probabilistic Approach ◽  
Modern Human ◽  
Peptide Sequence ◽  
Human Species ◽  
Interpretation Errors ◽  
Species Specific ◽  
Sequence Errors ◽  
Specific Peptide

AbstractProteogenomics is an increasingly common method for species identification as it allows for rapid and inexpensive interrogation of an unknown organism’s proteome—even when the proteome is partially degraded. The proteomic method typically uses tandem mass spectrometry to survey all peptides detectable in a sample that frequently contains hundreds or thousands of proteins. Species identification is based on detection of a small numbers of species-specific peptides. Genetic analysis of proteins by mass spectrometry, however, is a developing field, and the bone proteome, typically consisting of only two proteins, pushes the limits of this technology. Nearly 20% of highly confident spectra from modern human bone samples identify non-human species when searched against a vertebrate database—as would be necessary with a fragment of unknown bone. These non-human peptides are often the result of current limitations in mass spectrometry or algorithm interpretation errors. Consequently, it is difficult to know if a “species-specific” peptide used to identify a sample is actually present in that sample. Here we evaluate the causes of peptide sequence errors and propose an unbiased, probabilistic approach to determine the likelihood that a species is correctly identified from bone without relying on species-specific peptides.

scholarly journals Development of an Inducible Secretory Expression Vector and Host System for High Yield Production of Recombinant Protein

2021 ◽  
Author(s):  
Sunghyun Yoon ◽  
Keun Seok Seo ◽  
Nogi Park ◽  
Jaime Rutter ◽  
Justin A Thornton ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Recombinant Proteins ◽  
Large Scale ◽  
Expression Vector ◽  
Peptide Sequence ◽  
Host Strain ◽  
Vector System ◽  
Secretory Expression ◽  
Gram Positive

Abstract BackgroundEscherichia coli has been the most widely used recombinant protein expression system due to the availability of various protein expression vectors and ease of genetic manipulation. However, recombinant proteins expressed in E. coli are often contaminated with lipopolysaccharide (LPS) highly toxic to humans and must be removed from FDA-approved biologics, a process which requires extensive and expensive procedures. Gram-positive bacteria possess a single layer of cytoplasmic membrane free of LPS which make it ideal for producing recombinant protein. However, a lack of inducible protein expression systems limits a large-scale protein production in Gram-positive bacteria.ResultsThe HptARS is a three-component regulatory system in Staphylococcus aureus which senses extracellular glucose-6-phosphate and activates the uhpT gene promoter to facilitate uptake of extracellular G6P. To construct an inducible and secretory protein expression vector system, the promoter of the uhpT gene and the N-terminal signal peptide sequence of the hlb gene was fused in-frame with a C-terminal 6x-histidine sequence. For constitutive expression, we generated S. aureus expression host strain lacking the uhpT gene which could not uptake extracellular G6P, resulting in constitutive activation of HptARS system. With this newly established expression vector system and host strain, we demonstrated large-scale production of biologically active and highly pure staphylococcal leukotoxin E. ConclusionExtracellular expression of recombinant protein in LPS-free bacteria has a tremendous advantage in industrial production of FDA-approved biologics. Our newly established inducible and secretory expression vector system and S. aureus host strain will be useful to produce recombinant proteins for vaccine applications and other industrial purposes.

scholarly journals The remarkable solubility-enhancing power of Escherichia coli maltose-binding protein

2016 ◽  
Vol 62 (3) ◽  
pp. 377-382
Author(s):  
David S Waugh
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Inclusion Bodies ◽  
Binding Protein ◽  
Maltose Binding Protein ◽  
Review Article ◽  
Biologically Active ◽  
Active Protein ◽  
Inactive Form ◽  
Solubility Enhancers

A common problem encountered during the production of recombinant proteins, particularly in bacteria, is their tendency to accumulate in an insoluble and inactive form (i.e., as inclusion bodies). Although sometimes it is possible to convert the aggregated material into native, biologically active protein, this is a time-consuming, costly, and uncertain undertaking. Consequently, a general means of circumventing the formation of inclusion bodies is highly desirable. During the 1990s, it was serendipitously discovered that certain highly soluble proteins have the ability to enhance the solubility of their fusion partners, thereby preventing them from forming insoluble aggregates. In the ensuing years, Escherichia coli maltose-binding protein (MBP) has emerged as one of the most effective solubility enhancers. Moreover, once rendered soluble by fusion to MBP, many proteins are able to fold into their biologically active conformations. This brief review article focuses on our current understanding of what makes MBP such an effective solubility enhancer and how it facilitates the proper folding of its fusion partners.

The integration of a Stat3 specific peptide aptamer into the thioredoxin scaffold protein strongly enhances its inhibitory potency

2011 ◽  
Vol 5 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Hannah Schöneberger ◽  
Astrid Weiss ◽  
Boris Brill ◽  
Natalia Delis ◽  
Corina Borghouts ◽  
...  
Keyword(s):  
Amino Acids ◽  
Recombinant Protein ◽  
Protein A ◽  
Genetic Selection ◽  
Scaffold Protein ◽  
Peptide Sequence ◽  
Yeast Cells ◽  
Tumor Cell Death ◽  
Dimerization Domain ◽  
Specific Peptide

AbstractWe are characterizing peptides which are able to interact with functional domains of oncoproteins and thus inhibit their activity. The yeast two-hybrid system was used to derive a peptide sequence which specifically interacts with the dimerization domain of the transcription factor Stat3. The activated form of Stat3 is required for the survival of many transformed cells and Stat3 inhibition can cause tumor cell death. The genetic selection of specific peptide sequences from random peptide libraries requires the integration into a scaffold protein and the expression in yeast cells. The scaffold protein, a variant of the human thioredoxin protein, has previously been optimized and also allows for effective bacterial expression of the recombinant protein and the cellular uptake of the purified, recombinant protein. We investigated the contributions of the scaffold protein to the inhibitory properties of rS3-PA. For this purpose we compared rS3-PA in which the ligand peptide is embedded within the thioredoxin scaffold protein with a minimal Stat3-interacting peptide sequence. sS3-P45 is a synthetic peptide of 45 amino acids in length and consists only of the Stat3-binding sequence of 20 amino acids, a protein transduction domain (PTD) and a Flag-tag. Both, the recombinant rS3-PA of 19.3 kDa and the synthetic sS3-P45 of 5.1 kDa, were taken up into the cytoplasm of cells by the PTD-mediated transduction process, inhibited Stat3 target gene expression and caused the death of Stat3-dependent tumor cells. Stat3-independent normal cells were unaffected. rS3-PA effectively inhibited Stat3 function at 2 μM, however, sS3-P45 was required at a concentration of 100 μM to exert the same effects. The more potent action of rS3-PA is most probably due to a conformational stabilization of the Stat3-interacting peptide in the context of the scaffold protein.

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.

Sign in / Sign up

Export Citation Format

Share Document