Optimization of Human Serum Albumin Periplasmic Localization in Escherichia coli Using In Silico Evaluation of Different Signal Peptides

International Journal of Peptide Research and Therapeutics - Signal peptides (SPs) are essential tools to keep folding and function of recombinant proteins which are folded by disulfide bonds....     

In Silico Study of Different Signal Peptides to Express Recombinant Glutamate Decarboxylase in the Outer Membrane of Escherichia coli

International Journal of Peptide Research and Therapeutics - Gamma amino butyric acid (GABA) is used as drugs, food ingredients, and dietary supplements. l-glutamate is converted to GABA by the...     

In Silico Analysis for Determination and Validation of Iron-Regulated Protein from Escherichia coli

International Journal of Peptide Research and Therapeutics - The iron ion is an essential element in biological processes. Many of biological activities in cells, such as peroxide reduction,...     

Experimental Analysis of E2BB (LTIIb) Signal Peptide in Secretory Production of Reteplase in Escherichia coli

International Journal of Peptide Research and Therapeutics - Recombinant reteplase is the truncated form of tissue plasminogen activator. Signal peptides play a pivotal role in the secretion of...     

In silico Analysis of Different Signal Peptides for the Excretory Production of Recombinant NS3-GP96 Fusion Protein in Escherichia coli

International Journal of Peptide Research and Therapeutics - Escherichia coli is one of the simplest hosts which is widely being used to express heterologous proteins. However, without appropriate...     

Evaluation of Selected Flavonoids as Antiangiogenic,Anticancer, and Radical Scavenging Agents: An Experimental and In Silico Analysis

Developing antiangiogenic agents using natural products has remained a significant hope in the mainstream of anticancer research. In the present investigation series of flavonoids possessing di-, tri-, tetra-, and penta-hydroxy substitutions were evaluated as antiangiogenic agents using in vivo choriallantoic membrane model. The MTT-based cytotoxicity against selected cancer cell lines was carried out to determine the anticancer potential. The kinetics of free radical scavenging activities of these compounds was demonstrated using 2,2-diphenyl-1-picryl hydrazine (DPPH) and superoxide anion radicals (SORs). To understand the possible antiangiogenic mechanism, the selected flavonoids were docked in silico onto the proangiogenic peptides such as vascular endothelial growth factor (VEGF), hypoxia inducible factor (HIF-1α), and vascular endothelial growth factor receptor-2 (VEGFR2) from human origin. The results of the study shows that amongst the tested flavonoids, genistein (87.1%), kaempferol, (86.3%), and quercetin (84.7%) were found to be effective inhibitors of angiogenesis in CAM model. The antiangiogenic, cytotoxic, and antioxidant activities are discussed in light of structure–activity relationship using in silico approach and other drug-related properties were also calculated using BioMed CAChe V. 6.1.10. The results of the present study focus the isoflavone genistein, kaempferol, and quercetin as lead molecules for designing novel anti-tumor/antioxidant agents targeting angiogenesis.     

Expression of Erwinia chrysanthemi ENA49 uvr gene in Escherichia coli K12 cells

The uvrA gene of Erwinia chrysanthemi ENA49 similar to uvrA gene of Escherichia coli K12 has been cloned in vivo in Escherichia coli AB1886 uvrA6 cells using the plasmid pULB113 (RP4mini Mu). The presence of pULB113 carrying uvrA gene of Erwinia in Escherichia coli K12 uvrA- cells resulted in suppression of this mutation while uvrB and uvrC are not suppressed by this locus. The genetic control of excision repair of UV-damage in Erwinia chrysanthemi ENA49 is concluded to be similar to the one in Escherichia coli K12.     

重组蛋白在大肠杆菌周间腔的分泌表达

大肠杆菌是用于生产重组蛋白的重要工程宿主菌。但是,要获得足够的正确折叠的蛋白还存在一定的缺陷,其中一种解决此问题的方法就是使重组蛋白分泌到大肠杆菌的周间腔里。在这篇综述中,主要讨论了使重组蛋白分泌表达至大肠杆菌周间腔的近期的研究进展。     

Secretory expression of active clostripain in Escherichia coli

In this study, the clostripain gene was modified and its signal sequence was replaced with that of penicillin G acylase (PGA). The core clostripain protein fused to the PGA signal peptide was also prepared. With regard to the expression of the clostripain precursors, the majority of clostripain activity was observed in the culture media, thereby indicating that both the clostripain signal peptide and the PGA signal peptide were recognized in the E. coli secretion pathway, and the precursors successfully matured into the active form. Otherwise, the activity was rather low when the core protein was expressed, which indicates that the clostripain pro-peptide is important in the formation of the active enzyme in E. coli. Enzyme activity reached a value of 3200U/L in CGY media for high expression. The recombinant clostripain and porcine carboxypeptidase B were used in the conversion of a proinsulin fusion protein into insulin. The leader peptide (LP) and the proinsulin C-peptide appeared to have been removed simultaneously, and the final cleavage product evidenced an HPLC retention time identical to that of the insulin standard, thereby implying that the clostripain specifically cleaved the arginine residues in the LP and in the C-peptide. We have also demonstrated the possibility that the recombinant clostripain might prove useful in the production of insulin from the proinsulin fusion protein.     

Secretory production of human leptin in Escherichia coli

Human leptin is a 16 kDa (146 amino acids) protein secreted from adipocytes and influences body weight homeostasis. In this study, human leptin was produced and secreted efficiently in Escherichia coli using a novel Bacillus sp. endoxylanase signal peptide. The endoxylanase signal sequence consisted of 28 amino acids (84 bp) was fused to the leptin structural gene. The fused gene was expressed using an inducible promoter (T7 or Trc) by adding 1 mM IPTG. Using T7 promoter in E. coli BL21(DE3), most of protein produced was in a premature form. Using the Trc promoter, which is weaker than T7, leptin was efficiently produced and secreted as a mature form (40% of total proteins) at 37 degrees C. However, most of leptin (about 90%) formed the inclusion bodies in the periplasmic space of E. coli. At 30 degrees C, ca. 90% of leptin was produced in a soluble form, but the total amount of leptin produced was 40% less than that obtained at 37 degrees C. When the periplasmic oxidoreductase of E. coli, DsbA, was co-expressed, 69% of the secreted leptin (26% of total proteins) was produced as soluble form at 37 degrees C without the decrease of the amount of leptin produced.     

Cloning and Expression in Escherichia coli of the Polysaccharide Depolymerase Associated with Bacteriophage-Infected Erwinia amylovora

The bacteriophage-encoded polysaccharide depolymerase produced in Erwinia amylovora has been cloned and expressed in Escherichia coli. The bacteriophage ERA103 genome was observed to consist of five EcoRI fragments, labeled as follows: A, 7.5 kilobases (kb); B, 5.0 kb; C, 2.7 kb; D, 2.1 kb; and E, 1.8 kb. A restriction map for ERA103 was also prepared. Each of the fragments were cloned into the positive-selection vector pOP203(A₂⁺) and pBR322.     

In Silico Evaluation of Food Derived Bioactive Peptides as Inhibitors of Angiotensin Converting Enzyme (ACE)

International Journal of Peptide Research and Therapeutics - Hypertension is declared as the major risk factor of cardiovascular diseases and stroke, and the leading cause of premature deaths. ACE...     

In Silico Identification of Multi-target Anti-SARS-CoV-2 Peptides from Quinoa Seed Proteins

International Journal of Peptide Research and Therapeutics - Peptides are promising antagonists against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). To expedite drug...     

In Silico Design,Synthesis, and In Vitro Evaluation of Novel Amphipathic Short Linear Peptides Against Clinically Relevant Bacterial Biofilms

Microbial biofilms are organized communities of cells that are associated with a wide spectrum of resistant and chronic infections that lead to the treatment failure. Accordingly, there is an urgent demand to create novel effective therapeutic drugs that can inhibit biofilm formation with new mechanisms of action to surmount the current escalating resistance. In this study, in silico hybrid model was utilized to develop three novel short linear peptides (4, 5, and 6) with potential biofilm inhibiting activities (scores?>?1.0). The peptides were composed of cationic and hydrophobic residues. They were synthesized using solid-phase strategy. Synthesized peptides were purified and characterized by reverse-phase high-performance liquid chromatography and matrix-assisted laser desorption/ionization spectroscopy, respectively. They were evaluated using in vitro assay as potential inhibitors of clinically relevant Gram-positive and Gram-negative biofilms. Peptide (4) with five positive charges at physiological pH (4 cationic moieties and W:R?=?1:4) showed activity against biofilms of Gram-positive strains (Staphylococcus epidermidis and Listeria monocytogenes). On the other hand, peptide (5) with six positive charges (5 cationic moieties and W:R?=?2:2) demonstrated activity against Gram-positive (S. epidermidis) and Gram-negative (Escherichia coli) biofilms. Interestingly, peptide (6), with seven positive charges (6 cationic moieties and W:R?=?2:5) revealed higher and broader spectrum of activity against biofilms of Gram-positive (S. epidermidis, S. aureus, L. monocytogenes) and Gram-negative (E. coli).

     

信号肽对酵母外源蛋白质分泌效率的影响

根据酵母表达系统在表达外源蛋白方面的独特优势,利用酵母表达系统高效分泌并纯化具有生物学活性的蛋白质,已受到广泛关注。信号肽在蛋白质的分泌中起着重要作用,可引导蛋白分泌至胞外,大大提高蛋白的表达量,在工业化生产外源蛋白的纯化工艺方面具有重要意义。该文将从酵母表达系统中对信号肽的选择、改造、偏爱密码子和增强子的应用等几个方面进行优化的探讨,以提高蛋白质在酵母系统中的分泌效率。     

Cloning and expression of the Erwinia chrysanthemi asparaginase gene in Escherichia coli and Erwinia carotovora

A genomic library of Erwinia chrysanthemi DNA was constructed in bacteriophage lambda 1059 and recombinants expressing Er. chrysanthemi asparaginase detected using purified anti-asparaginase IgG. The gene was subcloned on a 4.7 kb EcoRI DNA restriction fragment into pUC9 to generate the recombinant plasmid pASN30. The position and orientation of the asparaginase structural gene was determined by subcloning. The enzyme was produced at high levels in Escherichia coli (5% of soluble protein) and was shown to be exported to the periplasmic space. Purified asparaginase from E. coli cells carrying pASN30 was indistinguishable from the Erwinia enzyme on the basis of specific activity [660-700 units (mg protein)-1], pI value (8.5), and subunit molecular weight (32 X 10(3]. Expression of the cloned gene was subject to glucose repression in E. coli but was not significantly repressed by glycerol. Recombinant plasmids, containing the asparaginase gene, when introduced into Erwinia carotovora, caused increased synthesis of the enzyme (2-4 fold higher than the current production strain).     

CisSERS: Customizable In Silico Sequence Evaluation for Restriction Sites

High-throughput sequencing continues to produce an immense volume of information that is processed and assembled into mature sequence data. Data analysis tools are urgently needed that leverage the embedded DNA sequence polymorphisms and consequent changes to restriction sites or sequence motifs in a high-throughput manner to enable biological experimentation. CisSERS was developed as a standalone open source tool to analyze sequence datasets and provide biologists with individual or comparative genome organization information in terms of presence and frequency of patterns or motifs such as restriction enzymes. Predicted agarose gel visualization of the custom analyses results was also integrated to enhance the usefulness of the software. CisSERS offers several novel functionalities, such as handling of large and multiple datasets in parallel, multiple restriction enzyme site detection and custom motif detection features, which are seamlessly integrated with real time agarose gel visualization. Using a simple fasta-formatted file as input, CisSERS utilizes the REBASE enzyme database. Results from CisSERS enable the user to make decisions for designing genotyping by sequencing experiments, reduced representation sequencing, 3’UTR sequencing, and cleaved amplified polymorphic sequence (CAPS) molecular markers for large sample sets. CisSERS is a java based graphical user interface built around a perl backbone. Several of the applications of CisSERS including CAPS molecular marker development were successfully validated using wet-lab experimentation. Here, we present the tool CisSERS and results from in-silico and corresponding wet-lab analyses demonstrating that CisSERS is a technology platform solution that facilitates efficient data utilization in genomics and genetics studies.     

Antifungal Signature: Physicochemical and Structural In Silico Analysis of Some Antifungal Peptides

High throughput screening of antifungal peptide libraries have generated large number of information on peptide activities. However scientists are still struggling to explain the specific antifungal protein motifs resulting in their activities. So a thorough antifungal peptide optimization is required. To design and predict secondary structure of synthetic as well as natural antifungal peptide using in silico approaches, various parameters such as their sequences, physicochemical characterization, structures and functions should taken care to ensure a successful prediction. The primary interest of this study is to determine the properties that stabilize bonds of helices and coils of antifungal peptides along with their domains. Fifty different antifungal peptides have been analyzed in this study which were retrieved from uniprot and pdb databases and were characterized thoroughly using in silico tools. The present analysis showed that most of the antifungal peptides were hydrophobic in nature due to high content of nonpolar residues where as the functional domains were hydrophilic because of the presence of more polar residues. Many disulphide bonds were noticed in these peptides because of the presence of high cysteine residues which may be regarded as a positive factor for stability of linear helices and coils. Maximum number of random coils were observed in the domains of the studied peptides. The present study thus indicated that the peptidal domains of antifungal peptides contain structurally conserve signature though they are evolutionary diverse. Thus the present in silico models are able to guide the antifungal peptide design in a meaningful way.     

In Vitro and In Silico Analysis of Novel ACE-Inhibitory Bioactive Peptides Derived from Fermented Goat Milk

In the study, two Lactobacillus cultures i.e. L. casei (NK9) and L. fermentum (LF) were studied for their proteolytic activity, di and tripeptidase activity, ACE-inhibitory activity and peptides production under optimized growth condition from fermented goat milk (Capra aegagrus hircus). NK9 and LF were found to be a strong proteolytic culture with 2.0% rate of inoculation after 48 h. LF (10 kDa retentate) produced maximum peptides among all the retentates of the fermented goat milk. Goat milk fermented with NK9 (10 kDa permeates) exhibited peptide sequence i.e. AFPEHK which had ACE inhibitory activity, matched with goat milk protein databases of AHTPDB. However, L. casei (NK9) and L. fermentum (LF) could be explored for the production of ACE inhibitory peptides from fermented goat milk.