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1.
Objectives
There are a number of blockbuster monoclonal antibodies on the market used for the treatment of a variety of diseases. Although the formulation of many antibodies is achieved in ‘platform’ formulations, some are so difficult to formulate that it can result in an inability to develop a finished drug product. Further, a large number of antibody-inspired or-based molecules are now being developed and assessed for biotherapeutic purposes and less is understood around the required active protein drug concentrations, excipients and additives required in final product formulations.Results
We investigated the effect of formulation variables (pH, buffer composition, glycine and NaCl concentration, time and temperature of accelerated stability studies) on antibody solubility/aggregation and activity using a Plackett–Burman Experimental Design approach. We then used the findings from this study and applied these to the formulation of a single chain variable fragment (ScFv) molecule. Our data shows that prediction of ScFc stability from a model monoclonal antibody could be achieved although further formulation optimization was required. Mass spectrometry analysis confirmed changes to the mass and hence authenticity of both the model antibody and ScFv under formulation conditions that did not provide appropriate conditions for protection of the molecules.Conclusions
The role of the different formulation conditions on maintaining protein integrity is described and using mass spectrometry shows that protein integrity is compromised under particular conditions. The implications for predicting successful formulations for protein molecules is discussed and how antibody formulations could be used to predict formulation components for novel antibody based molecules.2.
Expression and purification of classical swine fever virus E2 protein from Sf9 cells using a modified vector 总被引:1,自引:0,他引:1
Objective
To develop a simple method for efficient expression of classical swine fever virus (CSFV) E2 protein.Results
The pFastBac HT B vector (pFastHTB-M1) was modified by adding a melittin signal peptide sequence. The E2 gene fragment without the transmembrane region was cloned into pFastHTB-M1. The modified vector has clear advantage over the original one, as evidenced by the purified recombinant E2 protein that was detected significantly by SDS-PAGE.Conclusions
The modified vector has the potential for large-scale production and easy purification of the CSFV E2 protein or other proteins of interests.3.
Background
The protein encoded by the gene ybgI was chosen as a target for a structural genomics project emphasizing the relation of protein structure to function.Results
The structure of the ybgI protein is a toroid composed of six polypeptide chains forming a trimer of dimers. Each polypeptide chain binds two metal ions on the inside of the toroid.Conclusion
The toroidal structure is comparable to that of some proteins that are involved in DNA metabolism. The di-nuclear metal site could imply that the specific function of this protein is as a hydrolase-oxidase enzyme.4.
Background
Intrinsically disordered proteins (IDPs) and regions (IDRs) perform a variety of crucial biological functions despite lacking stable tertiary structure under physiological conditions in vitro. State-of-the-art sequence-based predictors of intrinsic disorder are achieving per-residue accuracies over 80%. In a genome-wide study of intrinsic disorder in human genome we observed a big difference in predicted disorder content between confirmed and putative human proteins. We investigated a hypothesis that this discrepancy is not correct, and that it is due to incorrectly annotated parts of the putative protein sequences that exhibit some similarities to confirmed IDRs, which lead to high predicted disorder content.Methods
To test this hypothesis we trained a predictor to discriminate sequences of real proteins from synthetic sequences that mimic errors of gene finding algorithms. We developed a procedure to create synthetic peptide sequences by translation of non-coding regions of genomic sequences and translation of coding regions with incorrect codon alignment.Results
Application of the developed predictor to putative human protein sequences showed that they contain a substantial fraction of incorrectly assigned regions. These regions are predicted to have higher levels of disorder content than correctly assigned regions. This partially, albeit not completely, explains the observed discrepancy in predicted disorder content between confirmed and putative human proteins.Conclusions
Our findings provide the first evidence that current practice of predicting disorder content in putative sequences should be reconsidered, as such estimates may be biased.5.
Bing Lin Xu-Zhong Yang Xue-Wei Cao Tao-Zhu Zhang Fu-Jun Wang Jian Zhao 《Biotechnology letters》2017,39(1):71-78
Objective
To evaluate the anti-tumor effects of trichosanthin after fusion with a cell penetrating peptide, heparin-binding peptide (HBP), derived from human heparin-binding EGF-like growth factor (HB-EGF).Results
The fusion protein of trichosanthin-HBP was expressed in Escherichia coli BL21 and purified by Ni–NTA affinity chromatography. The HBP domain had no influence on the topological inactivation activity and N-glycosidase activity of trichosanthin. Trichosanthin-HBP significantly inhibited the growth of tested cancer cells which are impervious to trichosanthin. Tumor cell apoptosis and both the mitochondrial- and death receptor-mediated apoptotic signaling pathways induced by trichosanthin-HBP were more significant than those induced by trichosanthin in HeLa cells.Conclusion
HBP is an efficient intracellular delivery vehicle for trichosanthin and makes trichosanthin-HBP become a promising agent for cancer therapy.6.
Arshid Yousefi-Avarvand Mohsen Tafaghodi Saman Soleimanpour Farzad Khademi 《生物学前沿》2018,13(4):293-296
Background
Tuberculosis (TB) is a contagious infectious disease caused by Mycobacterium tuberculosis (Mtb). This disease with two million deaths per year has the highest mortality rate among bacterial infections. The only available vaccine against TB is BCG vaccine. BCG is an effective vaccine against TB in childhood, however, due to some limitations, has not proper efficiency in adults. Also, BCG cannot produce an adequately protective response against reactivation of latent infections.Objective
In the present study we will review the most recent findings about contribution of HspX protein in the vaccines against tuberculosis.Methods
Therefore, many attempts have been made to improve BCG or to find its replacement. Most of the subunit vaccines for TB in various phases of clinical trials were constructed as prophylactic vaccines using Mtb proteins expressed in the replicating stage. These vaccines might prevent active TB but not reactivation of latent tuberculosis infection (LTBI). A literature search was performed on various online databases (PubMed, Scopus, and Google Scholar) regarding the roles of HspX protein in tuberculosis vaccines.Results
Ideal subunit post-exposure vaccines should target all forms of TB infection, including active symptomatic and dormant (latent) asymptomatic forms. Among these subunit vaccines, HspX is the most important latent phase antigen of M. tuberculosis with a strong immunological response. There are many studies that have evaluated the immunogenicity of this protein to improve TB vaccine.Conclusion
According to the studies, HspX protein is a good candidate for development of subunit vaccines against TB infection.7.
Background
Proteins play fundamental and crucial roles in nearly all biological processes, such as, enzymatic catalysis, signaling transduction, DNA and RNA synthesis, and embryonic development. It has been a long-standing goal in molecular biology to predict the tertiary structure of a protein from its primary amino acid sequence. From visual comparison, it was found that a 2D triangular lattice model can give a better structure modeling and prediction for proteins with short primary amino acid sequences.Methods
This paper proposes a hybrid of hill-climbing and genetic algorithm (HHGA) based on elite-based reproduction strategy for protein structure prediction on the 2D triangular lattice.Results
The simulation results show that the proposed HHGA can successfully deal with the protein structure prediction problems. Specifically, HHGA significantly outperforms conventional genetic algorithms and is comparable to the state-of-the-art method in terms of free energy.Conclusions
Thanks to the enhancement of local search on the global search, the proposed HHGA achieves promising results on the 2D triangular protein structure prediction problem. The satisfactory simulation results demonstrate the effectiveness of the proposed HHGA and the utility of the 2D triangular lattice model for protein structure prediction.8.
9.
Background
Analysis of preferred binding regions of a ligand on a protein is important for detecting cryptic binding pockets and improving the ligand selectivity.Result
The enhanced sampling approach TAMD has been adapted to allow a ligand to unbind from its native binding site and explore the protein surface. This so-called re-TAMD procedure was then used to explore the interaction between the N terminal peptide of histone H3 and the YEATS domain. Depending on the length of the peptide, several regions of the protein surface were explored. The peptide conformations sampled during the re-TAMD correspond to peptide free diffusion around the protein surface.Conclusions
The re-TAMD approach permitted to get information on the relative influence of different regions of the N terminal peptide of H3 on the interaction between H3 and YEATS.10.
Yu Zhang Yue Tong Mingming Gao Cheng Luo Xiaoda Song Xiangdong Gao Wenbing Yao 《Biotechnology letters》2016,38(7):1115-1120
Objectives
To prepare recombinant tPep-(vascular endothelial growth factor) VEGF-B and assess its biological activity.Results
This new VEGF fusion protein was constructed using a targeting peptide and prepared using E.coli. The tPep-VEGF-B was refolded from inclusion bodies and purified using affinity chromatography. Its bioactivity was determined in vitro using proliferation assay and wounding healing assay, and in vivo in zebrafish. By using the optimized downstream process, recombinant tPep-VEGF-B can be obtained with a purity of >90 % and a yield of 80 mg protein/l culture medium. The refolded protein is highly effective in promoting cell migration in vitro and in enhancing angiogenesis in vivo.Conclusion
We have constructed a new VEGF fusion protein with potential therapeutic application in treating metabolic diseases.11.
Background
Many protein regions and some entire proteins have no definite tertiary structure, presenting instead as dynamic, disorder ensembles under different physiochemical circumstances. These proteins and regions are known as Intrinsically Unstructured Proteins (IUP). IUP have been associated with a wide range of protein functions, along with roles in diseases characterized by protein misfolding and aggregation.Results
Identifying IUP is important task in structural and functional genomics. We exact useful features from sequences and develop machine learning algorithms for the above task. We compare our IUP predictor with PONDRs (mainly neural-network-based predictors), disEMBL (also based on neural networks) and Globplot (based on disorder propensity).Conclusion
We find that augmenting features derived from physiochemical properties of amino acids (such as hydrophobicity, complexity etc.) and using ensemble method proved beneficial. The IUP predictor is a viable alternative software tool for identifying IUP protein regions and proteins.12.
13.
Ming-zhu Wang Zhuo-lin Qiu Xiang-Sheng Cai Jing-jing Li Miao-qin She Yuan-feng Xu Ying-song Wu 《Biotechnology letters》2017,39(10):1529-1535
Objective
To produce a recombinant spermatozoa antigen peptide using the E. coli: PhoA system on a protein chip for screening anti-sperm antibodies (ASA).Results
The purity of the recombinant spermatozoa antigen exceeded 95% after two-step purification, as assessed using SDS-PAGE and HPLC. The diagnostic performance of a protein chip coated with the recombinant antigen peptide was evaluated by examining ASA in 51 infertile patients in comparison with a commercial ELISA kit. The area under the receiver operating characteristic curve (AUC) was 0.944, which indicated that the protein chip coated with recombinant spermatozoa antigen peptide was consistent with ELISA for ASA detection.Conclusion
A recombinant spermatozoa antigen was expressed in the E. coli PhoA secretory expression system and its potential application for clinical ASA detection was validated.14.
Daria V. Dibrova Kirill A. Konovalov Vadim V. Perekhvatov Konstantin V. Skulachev Armen Y. Mulkidjanian 《Biology direct》2017,12(1):29
Background
The Clusters of Orthologous Groups (COGs) of proteins systematize evolutionary related proteins into specific groups with similar functions. However, the available databases do not provide means to assess the extent of similarity between the COGs.Aim
We intended to provide a method for identification and visualization of evolutionary relationships between the COGs, as well as a respective web server.Results
Here we introduce the COGcollator, a web tool for identification of evolutionarily related COGs and their further analysis. We demonstrate the utility of this tool by identifying the COGs that contain distant homologs of (i) the catalytic subunit of bacterial rotary membrane ATP synthases and (ii) the DNA/RNA helicases of the superfamily 1.Reviewers
This article was reviewed by Drs. Igor N. Berezovsky, Igor Zhulin and Yuri Wolf.15.
Dawei Jiang Yunchao Liu Aiping Wang Gaiping Zhang Guoyu Yang Yumei Chen Pengchao Ji Chang Liu Yapeng Song Yunfang Su Guoqiang Wang Jucai Wang Baolei Zhao Ruiguang Deng 《Biotechnology letters》2016,38(6):901-908
Objectives
To improve the expression of soluble IBDV VP2 protein by using different tagged vectors in Escherichia coli.Results
Fusion tags, Grifin, MBP, SUMO, thioredoxin, γ-crystallin, ArsC and PpiB, enhanced the expression and solubility of VP2 protein. The fusion proteins were purified by Ni–NTA chromatography, MBP-VP2 showed the highest purity about 90 %. After removing the MBP tag, VP2 self-assembled into virus-like particles, ~25 nm diam. Results from AGP suggested the recombinant IBDV VP2 protein identified by reference serum like IBDV.Conclusion
All the seven tags enhanced the expression and solubility of IBDV VP2 protein. The recombinant protein self-assembly into virus like particles and possess antigenicity as reference IBDV.16.
Background
Protein complexes play an important role in biological processes. Recent developments in experiments have resulted in the publication of many high-quality, large-scale protein-protein interaction (PPI) datasets, which provide abundant data for computational approaches to the prediction of protein complexes. However, the precision of protein complex prediction still needs to be improved due to the incompletion and noise in PPI networks.Results
There exist complex and diverse relationships among proteins after integrating multiple sources of biological information. Considering that the influences of different types of interactions are not the same weight for protein complex prediction, we construct a multi-relationship protein interaction network (MPIN) by integrating PPI network topology with gene ontology annotation information. Then, we design a novel algorithm named MINE (identifying protein complexes based on Multi-relationship protein Interaction NEtwork) to predict protein complexes with high cohesion and low coupling from MPIN.Conclusions
The experiments on yeast data show that MINE outperforms the current methods in terms of both accuracy and statistical significance.17.
Neha Dhami Drupad K. Trivedi Royston Goodacre David Mainwaring David P. Humphreys 《Metabolomics : Official journal of the Metabolomic Society》2018,14(10):136
Introduction
Mammalian cells like Chinese hamster ovary (CHO) cells are routinely used for production of recombinant therapeutic proteins. Cells require a continuous supply of energy and nutrients to sustain high cell densities whilst expressing high titres of recombinant proteins. Cultured mammalian cells are primarily dependent on glucose and glutamine metabolism for energy production.Objectives
The TCA cycle is the main source of energy production and its continuous flow is essential for cell survival. Modulated regulation of TCA cycle can affect ATP production and influence CHO cell productivity.Methods
To determine the key metabolic reactions of the cycle associated with cell growth in CHO cells, we transiently silenced each gene of the TCA cycle using RNAi.Results
Silencing of at least four TCA cycle genes was detrimental to CHO cell growth. With an exception of mitochondrial aconitase (or Aco2), all other genes were associated with ATP production reactions of the TCA cycle and their resulting substrates can be supplied by other anaplerotic and cataplerotic reactions. This study is the first of its kind to have established key role of aconitase gene in CHO cells. We further investigated the temporal effects of aconitase silencing on energy production, CHO cell metabolism, oxidative stress and recombinant protein production.Conclusion
Transient silencing of mitochondrial aconitase inhibited cell growth, reduced ATP production, increased production of reactive oxygen species and reduced cell specific productivity of a recombinant CHO cell line by at least twofold.18.
19.
Eriko Tomitsuka Katsura Igai Kiyoshi Tadokoro Ayako Morita Jun Baba Wataru Suda Andrew R. Greenhill Paul F. Horwood Kevin W. Soli Peter M. Siba Shingo Odani Kazumi Natsuhara Hidetoshi Morita Masahiro Umezaki 《Metabolomics : Official journal of the Metabolomic Society》2017,13(9):105
Introduction
Adequate amount of proteins from foods are normally needed to maintain muscle mass of the human body. Although protein intakes of Papua New Guinea (PNG) highlanders are less than biologically adequate, protein deficiency related disorders have rarely been reported. It has been postulated that gut microbiota play a role in such low-protein-adaptation.Objective
To explore underlying biological mechanisms of low-protein adaptation among PNG highlanders by investigating metabolomic profiles of faecal water and urine.Methods
We performed metabolome analysis using faecal water extracted from faecal samples of PNG highlanders, PNG non-highlanders and Japanese subjects. We paid special attention to amino acids and other metabolites produced by gut microbiota, as well as to metabolites involved in nitrogen recycling in the human gut.Results
Our results indicated that amino acid levels were higher in faecal water from PNG highlanders than PNG non-highlanders, but amino acid levels did not differ between PNG highlanders and Japanese subjects. Among PNG highlander samples, amino acid levels tended to be higher in those who consumed less protein.Conclusion
We speculated that a greater proportion of urea was excreted to the intestine among the PNG highlanders than other groups, and that the urea was used for nitrogen salvage. Intestinal bacteria are essential for producing ammonia from urea and also for producing amino acids from ammonia, which is a key process in low-protein adaptation. Profiling the gut microbiota of PNG highlanders is an important avenue for further research into the mechanisms of low-protein adaptation.20.
Marine Goux Amina Fateh Alain Defontaine Mathieu Cinier Charles Tellier 《Biotechnology letters》2016,38(5):767-772