Faculty of Science

  Initial Bioinformatic Investigation
Using Bioinformatic Tools to Strategically Design Expression/Purification Projects

Dr. Nurit Kleinberger-Doron

• ExPASy Proteomics Server
• SRS Available Analysis Tools .
• Bioinformatics, Genomics, Proteomics, Biotechnology and Molecular Biology Directory
• MyBio - the biologist's wiki workbench
• GenomeWeb -  (check the Proteins and Nucleic Acids sections)
• ONLINE ANALYSIS TOOLS

• NAR (Nucleic Acids Research) Database Categories List
• Toolbox compiled by the Bioinformatics & Biological Computing Unit, Weizmann Institute of Science.

Preliminary Search Using Keywords

• Entrez (NCBI) - A text-based search and retrieval system used at NCBI for the major databases, including PubMed, Nucleotide and Protein Sequences, Protein Structures, Complete Genomes, Taxonomy, and others.
• SRS (EBI) (Expasy) (Public SRS servers worldwide) - Sequence Retrieval System. List of SWISS-PROT keywords with strong correlation to sub-cellular localisation.
• Genecards - A database of human genes, their products and their involvement in diseases.
• Brenda - The Comprehensive Enzyme Information System.
• UCSC Proteome Browser

• Blast (Which BLAST tool do I use for different searches?)
• Fasta (EBI, U Virginia)
• Multiple alignment - ClustalW - EBI ,  T Coffee + M Coffee(CNRS, SIB) [T-Coffee: A novel method for fast and accurate multiple sequence alignment. Notredame C, Higgins DG, Heringa J. J Mol Biol. 2000 Sep 8;302(1):205-17.] M Coffee
• Pairwise alignments - Emboss Pairwise Alignment Algorithms ,   Lalign (Embnet),    Blast 2 sequences.

Secondary Structure of mRNA

• A web interface to the RNAfold programm (Part of the Vienna RNA Pasckage)
• RNA Folding Server - Zucker group.
• RNA secondary structure prediction

Codon Usage and Translation Frames

• Rare Codon Caltor - For expression in E-coli
• Rare Codon Calculator (RaCC)  - For expression in E-coli (defines less codons as rare, less "false positive" more "false negative")
• Search of rare codons in nucleotide sequence - For expression in 6 organisms.
• Optimizer - optimizes the codon usage of a DNA sequence to increase its expression level.
• Translation tools allowing change of genetic code: Transeq, DNA to protein translation , another DNA to protein translation , DNA aligned protein translation , Virtual Ribosome - version 1.1. Examine the different genetic codes.
• Translate a DNA Sequence - paste a nucleic acid sequence and obtain graphical and textual depictions of its possible translations (various ORFs) in all 6 reading frames.
• A Graphical Codon Usage Analyser.
• Codon usage database .

Alternative Splicing

• Known information - Gene database
• Alternative Splicing and Transcript Diversity (EBI)
• ASPIC (detects the exon-intron structure of a gene by comparing its genomic sequence to the related cluster of ESTs)
• ASSP
• SpliceNest
   

Motifs and Repeats in  Proteins

• Interpro - A database of protein families, domains and functional sites in which identifiable features found in known proteins can be applied to unknown protein sequences.
• CDD[Conserved Domain Database] - a collection of sequence alignments and profiles representing protein domains conserved in molecular evolution. (NCBI) [also appears as part of Blast output]. Run a BLAST search against the CDD.
• ELM - Eukaryotic Linear Motif Resource for Functional Sites in Proteins.
• Prosite - A database of protein families and domains. It consists of biologically significant sites, patterns and profiles that help to reliably identify to which known protein family (if any) a new sequence belongs.
• ProTeUs - (PROtein TErminUS) - a tool for the identification of short linear signatures in protein termini. (About)
• QuasiMotiFinder - A server for the identification of motifs and signature-like patterns in protein sequences (based on Prosite and multiple alignments)
• Motif Search - for DNA or protein sequences.
• Radar - Rapid Automatic Detection and Alignment of Repeats in protein sequences.
• SAPS - Statistical Analysis of Protein Sequences.
• Additional sites (Expasy list).

Physicochemical properties

• Protparam - Physico-chemical parameters of a protein sequence (amino-acid and atomic compositions, pI, extinction coefficient, etc.)
• Protein Calculator - Generates molecular weight information (including scanning mass spectrometry results), estimated charges (including pI estimation), uv absorption coefficients, crystallographic solvent content percentage and Vm, and counts atoms and residues based on the protein sequence.
• EMBOSS   Pepinfo/Pepwindow/Pepstats .
• ProtScale (several hydrophobicity scales)

• Antigenicity
• Emboss - Antigenic (England,) - finds antigenic sites in proteins.
• Prediction of antigenic peptides

• Protein Solubility
• Recombinant Protein Solubility Prediction - Predicts protein solubility assuming the protein is being overexpressed in Escherichia coli. (Based on: Wilkinson DL, Harrison RG., Predicting the solubility of recombinant proteins in Escherichia coli., Biotechnology (N Y). 1991 May;9(5):443-8.)
• FodIndex -  predicts whether a given protein sequence is intrinsically unfolded, based on the average residue hydrophobicity and net charge of the sequence. Look at additional prediction tools for disordered proteins.
• Refold - identifying optimal conditions and methodology for refolding.

Protein Turnover

• PESTfind - Polypeptide sequences enriched in Proline (P), glutamic acid (E), serine (S) and threonine (T) target proteins for rapid destruction. PESTfind produces a score ranging form about -50 to +50. By definition, a score above zero denotes a possible PEST region, but a value greater than +5 sparks real interest.
• Destruction Box (D box) Finder - characterizes some proteins destined to proteolysis by ubiquitin and the 26S proteasome pathway.
• Protparam - references to N-end rule (scroll down)
• I-Mutant2.0 - a tool for predicting protein stability upon single site mutation.

Proteolytic Cleavage

• Peptide Cutter - predicts potential protease and cleavage sites and sites cleaved by chemicals in a given protein sequence.
• Protease database of: E coli,
• MEROPS the peptidase database [Various search options.]
• Classification of peptidase families and index of peptidase entries in Swiss-Prot [Alan Barrett and Neil Rawlings]
• InBase - The Intein Database (NEB). Inteins are self-catalytic protein splicing elements. Blast your sequence against InBase. (Read about InBase)
• ProP - predicts arginine and lysine propeptide cleavage sites in eukaryotic protein sequences.

Co- and Post-translational Modifications (A short summary)

      Phosphorylation

• Netphos - Predicts serine, threonine and tyrosine phosphorylation sites in eukaryotic proteins.
• KinasePhos - computes the location of the phosphorylation sites and the corresponding catalytic protein kinases.
• NetPhosK - produces predictions of kinase specific eukaryotic protein phosphoylation sites. Currently NetPhosK covers the following kinases: PKA, PKC, PKG, CKII, Cdc2, CaM-II, ATM, DNA PK, Cdk5, p38 MAPK, GSK3, CKI, PKB, RSK, INSR, EGFR and Src.
• Scansite - searches for motifs within proteins that are likely to be phosphorylated by specific protein kinases or bind to domains such as SH2 domains, 14-3-3 domains or PDZ domains.
• GPS: Group-based Phosphorylation Scoring Method - in-silico prediction of phosphorylation sites of specific kinases.
• Phosphorylation Site Database - Phosphorylation in prokaryotes.


   Glycosylation

• YinOYang - Predicts O-ß-GlcNAc attachment sites in eukaryotic protein sequences.
• NetOGlyc  - Predicts mucin type GalNAc O-glycosylation sites in mammalian proteins.
• NetNGlyc - Predicts N-Glycosylation sites in human proteins.
• DictyOGlyc  - Predicts GlcNAc O-glycosylation sites in Dictyostelium discoideum proteins.


Addition of Lipid Moieties

• NMT - Predicts N-terminal N-Myristoylation by MyristoylCoA:Protein N-Myristoyltransferase.
• Myristoylator - predicts N-terminal myristoylation of proteins by neural networks.
• PrePS - Prenylation Prediction Suite (additional details).
• CSS-Palm - palmitoylation sites prediction
• GPI lipid anchor predictor - animals, plants.
• Detection/prediction of GPI cleavage site (GPI-anchor) in a protein
• GPI-SOM: Identification of GPI-anchor signals.


S-S bonds

• CYSPRED - Predicts cysteins that are likely to be partners in cysteine bridges. (Program described in: Fariselli P, Riccobelli P, Casadio R PROTEINS(1999) 36:340-346)
• DCON - Predictor of Disulfide Connectivity in Proteins.
• Disulfind - Cysteines Bonding State and Connectivity Predictor.
• Dianna - Cysteine state and Disulfide Bond partner prediction.
• CysView -
• GDAP - disulfide bond prediction by sequence to structure mapping


Others

• The Sulfinator - Predicts tyrosine sulfation sites in protein sequences
• NetAcet - predicts substrates of N-acetyltransferase A (NatA). The method was trained on yeast data but  it obtains similar performance values on mammalian substrates acetylated by NatA orthologs.
• ProMoST: Protein Modification Screening Tool -  calculates accurate MWT and pI values from proteins and presents putative 2-D gels.
• Additional sites of interest (Expasy list,  MolBiol.Net list)

Sub-cellular Localization and Signal Peptides

Subcellular Compartments

• psort - Programs for subcellular localization prediction (eukaryotic sequences, plant and Gram-positive bacterial sequences, Gram-negative bacterial sequences)
• SoftBerry Protein Location Finding - offers different programs for animal/fungi, plant and bacterial proteins.
• ESLPred - prediction of subcellular localization of proteins.
• TargetP Server - Predicts the subcellular location of eukaryotic protein sequences. The subcellular location assignment is based on the predicted presence of any of the N-terminal presequences chloroplast transit peptide (cTP), mitochondrial targeting peptide (mTP) or secretory pathway signal peptide (SP).
• PLOC - protein localization prediction (plants, fungi, animals).
• MultiLoc/TargetLoc (Tubingen University)
• LocTarget - from Columbia University Bioinformatics Center
• LOCSVMpsi - eukaryotic protein subcellular LOCalization based on SVM and PSI-blast
• LOCtree - a eukaryotic and prokaryotic localization prediction tool available at the CUBIC site.
• Proteome Analyst Specialized Subcellular Localization Server .
• SubLoc - Prediction of protein subcellular localization (contains less subcellular targets than other programs, and so may bias results)
• BLSTM-LOC Protein Localization Prediction Server (eukaryotes: plants vs. non plants)
• SecretomeP -  Prediction of non-classical and leaderless protein secretion. Produces ab initio predictions of non-classical i.e. not signal peptide triggered protein secretion. The method queries a large number of other feature prediction servers to obtain information on various post-translational and localizational aspects of the protein, which are integrated into the final secretion prediction. (Paper)
• Golgi transmembrane predictor - predicts Golgi membrane proteins based on their transmembrane domains. This prediction method is only valid for Type II transmembrane proteins, and output from the method is simply predicted to be Golgi localised or predicted to transit through the Golgi (post-Golgi localisation).
• PTS1 predictor - predicts the peroxisomal targeting signal 1.

• ipsort - predicts whether a sequence contains a Signal Peptide (SP), Mitochondrial Targeting Peptide (mTP), or Chloroplast Transit Peptide (cTP).
• SignalP - Predicts the presence and location of signal peptide cleavage sites in amino acid sequences from different organisms (Gram-positive prokaryotes, Gram-negative prokaryotes, and eukaryotes).
• Sigcleave - Reports protein signal cleavage sites.
• SIGFIND - Signal Peptide Prediction Server (Eukaryotes)
• Signal Peptide Prediction (Leeds University)
• SPEPLip - Predictor of Signal Peptide and Lipoprotein Cleavage Sites in Proteins
• LipoP - prediction of lipoproteins and signal peptides in Gram negative bacteria
• PredictNLS - analysis and determination of Nuclear Localization Signals
• NetNES - predicts leucine-rich nuclear export signals (NES) in eukaryotic proteins.
• ChloroP - Prediction of chloroplast transit peptides
• MitoProt - Prediction of mitochondrial targeting sequences.
• Predotar - Prediction of mitochondrial and plastid targeting sequences.
• SecretomeP - predicts non-classical i.e. not signal peptide triggered protein secretion in eukaryotes. The method queries a large number of other feature prediction servers to obtain information on various post-translational and localizational aspects of the protein, which are integrated into the final secretion prediction.
• TatP - predicts the presence and location of Twin-arginine signal peptide cleavage sites in bacteria.
• ProP - predicts arginine and lysine propeptide cleavage sites in eukaryotic protein sequences.

• List of SWISS-PROT keywords with strong correlation to sub-cellular localisation.

Protein-Protein Interactions

• STRING - a database of known and predicted protein-protein interactions. The interactions include direct (physical) and indirect (functional) associations; they are derived from four sources:  1. Genomic Context  2. High-throughput Experiments  3. (Conserved) Coexpression  4. Previous Knowledge .
• IntAct - all interactions are derived from literature curation or direct user submissions
• HGPRD (Human Protein Reference Database) - examine the sections: "interactions" & "PTMs and Substrates".
• DIP - Database of interacting proteins.
• MINT - Molecular Interactions Database.
• Bind - Biomolecular Interaction Network Database.
• iPfam - describes domain-domain interactions that are observed in PDB entries.
• InterDom - a database of putative interacting protein domains derived from multiple sources, ranging from domain fusions (Rosetta Stone), protein interactions (DIP and BIND), protein complexes (PDB), to scientific literature (MEDLINE).
• Additional sites (list of the UK Human Genome Mapping Project Resource Centre,  Finley Lab list)

• KEGG Pathway Database
• Reactome

Search for known structures using:

• PDB - RCSB protein data bank.
• MMDB - Entrez structures (molecular modeling database)
• MSD - EBI structures(macromolecular structures database)
• OCA - provides rich content annotation on structure and function, generating dynamic links to several external sources.
• PDBSum - is a pictorial database providing an at-a-glance overview of every macromolecular structure deposited in the PDB. It provides schematic diagrams of the molecules in each structure and of the interactions between them.
• iPfam - describes domain-domain interactions that are observed in PDB entries.

Structure & Function Predictions
 

Function Prediction

• ProtFun - predicts  protein function from sequence. The method queries a large number of other feature prediction servers to obtain information on various post-translational and localizational aspects of the protein, which are integrated into final predictions of the cellular role, enzyme class (if any), and selected Gene Ontology categories of the submitted sequence; Paper 1; Paper 2; A short presentation;
• GeneQuiz Server - derives functional annotation for protein sequences and provides supporting evidence, including family alignments.
• SVMProt: Protein Functional Family Prediction
• ConSEq - A server for the identification of functionally and structurally important residues in protein sequences without known structures.
• ConSurf - Server for the identification of functional regions in proteins with known structures.

• PsiPred - Protein Structure Prediction Server
• Proteus2 - bundles signal peptide identification, transmembrane helix prediction, transmembrane beta-strand prediction, secondary structure prediction (for soluble proteins) and homology modeling (i.e. 3D structure generation) into a single prediction pipeline.
• JPred3 - a secondary structure prediction server powered by the Jnet algorithm.
• PredictProtein or PredictProtein - offers the following: generation of multiple sequence alignments (MaxHom) , detection of functional motifs (PROSITE), detection of composition-bias (SEG), detection of protein domains (PRODOM), fold recognition by prediction-based threading (TOPITS), predictions of: secondary structure (PHDsec, and PROFsec), residue solvent accessibility (PHDacc, and PROFacc), transmembrane helix location and topology (PHDhtm, PHDtopology), protein globularity (GLOBE), coiled-coil regions (COILS), cysteine bonds (CYSPRED), structural switching regions (ASP)
• Expasy tools

Disordered Proteins

• DisProt database
• DisProt list of prediction tools

Topology Prediction

a helices

• Psipred - You may select one of three prediction methods to apply to your sequence: PSIPRED - a highly accurate method for protein secondary structure prediction, MEMSAT - a widely used transmembrane topology prediction method and GenTHREADER - a sequence profile based fold recognition method.
• TMHMM - Prediction of transmembrane helices in proteins,  nice graphics
• Phobius - a combined transmembrane topology and signal peptide predictor.
• SOSUI - Predicts transmembrane helices in proteins and includes the helical wheels in the graphic presentation. Checks for presence of signal peptide to avoid the risk of signal peptides being predicted as putative TM as well.
• BPROMPT - BayesianPRedictionOfMembraneProteinTopology. Uses a Bayesian Belief Network to combine the results of other prediction methods, providing a more accurate consensus prediction. Topology predictions

with accuracies of 70% for prokaryotes and 53% for eukaryotes were achieved. (Abstract of paper)
 
• Expasy's tools for topology prediction.

• ConBBPRED: Consensus Prediction of TransMembrane Beta-Barrel Proteins (about)
• PROFtmb - a prediction service for Bacterial Transmembrane Beta Barrels
• TMB-Hunt - a web server to screen sequence sets for transmembrane beta-barrel proteins.

• Helical wheel (Rutgers)
• Helical wheel
• Helical wheel
• Print a helical wheel

• Suggest an expression system - developed in Weizmann Institute of Sciences. SuggestES takes the protein sequence you provide and scans a large database with protein sequences with known results for different expression systems. At the time of generating a suggestion, suggestES takes into consideration several parameters:
1. Similarity: how similar is your sequence to the existing data in the database?. The expression systems used on sequences similar to yours are preferred when creating the list of suggestions.
2. Recentness: how recently was used a given expression system?. The older the record of the usage of a given expression system, the less this system will influence the final result. This will provide visibility to recently appearing system.
3. Frequency: how frequently a given expression system has been used?

This site is maintained by Dr. Nurit Doron . Your comments are most welcome.

entries since November 2003

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