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Historically, some important disease states were identified as being caused by the lack of an important protein, or the presence of a dysfunctional mutated form of a protein.
- For example, diabetes, types of dwarfism and hemophilia were found to be due to deficiencies in insulin, growth hormoneand clotting factor VIII, respectively.
These diseases could be treated by injecting supplemental doses of purified, or partially purified, preparations of these proteins.
- These proteins were isolated from natural materials, e.g. pig (insulin), human cadaver pituitaries (human growth hormone) or blood fractions pooled from normal donors (factor VIII).
- In most cases, even if the protein was found in relatively abundant supply, the cost of production was substantial.
More often than not, interesting bioactive properties were associated with proteins which could be isolated only in minute quantitites(e.g. the blood clot dissolving protein tissue plasminogen activator).
Also, non-human proteins typically elicited an immune response when injected into humans, thus the human form of a protein was the only useful form.
- If the protein were not readily available from blood, or urine, it would prove impractical to obtain adequate starting materialfor production.
- Unfortunately, if the material were derived from human sources, the possibility existed for the spread of human disease (e.g. hepatitis and the AIDS virus).
If the genetic information for these proteins could be isolated, and then transcribed and translated in an easily scaleable biological system, potentially large amounts of protein could be obtained - and hopefully, relatively cheaply.
With the development of "molecular biology", i.e.
- the structure of DNA,
- the elucidation of the genetic code,
- the identification of transcriptional promoters and ribosome binding sites,
- the isolation of restriction endonuclases,
- the identification of the origin of DNA replication
- the development of plasmids with selectable markers, and
- the culturing of E. coli,
the possibility existed in the mid 1970's to put it all together and produce relatively large amounts of any human protein for therapeutic use.
How would you go about the process of producing large amounts of some important human protein? (i.e. protein purification)
The starting point is typically an assay for a functionality of interest. For example, we may have a hemophiliac whose blood does not clot. However, we find that if we take a sample of his blood and add to it a small amount of blood from a "normal" individual, the hemophiliac's blood will now clot. This will be the basis for our assay.
Using this assay, we will fractionate normal blood using various means - chemical precipitation (with ethanol, or ammonium sulfate), and then various liquid chromatography steps, etc.
- Along the way we will follow where our clotting activity is going.
- Hopefully, at some point we will be unable to fractionate it further and will have a pure protein.
Once we have a pure protein we can begin to characterize it with regard to its amino acid sequence. From there we can ultimately get the gene for the protein and express it.
Figure 3.5.1: Protein production
N-terminal peptide sequence analysis
Polypeptides can be sequenced from their amino-terminus by automated procedures based upon the Edman degradation reaction:
Figure 3.5.2: Edman degradation
- Note that with Edman chemistry only the N-terminal residue is attacted and removed, the rest of the polypeptide remains intact after the reaction.
- The new amino terminal group (previously the second amino acid in the polypeptide chain) is now available for another round of reactions. Thus, the method can be automated.
- The amino acid side chain of the phenylthiohydantoin derivative can be identified using liquid chromatography. Modern amino acid sequencers can probably sequence on the order of two to three dozen cycles (amino acids) of a polypeptide.
- Note that the reaction requires a free amino group on the N-terminal of the protein. If the amino-terminal residue is methylated or formylated then the reaction will not proceed (and the polypeptide is said to have a "blocked" N-terminal).
C-terminal peptide sequence analysis
C-terminal peptide sequence analysis is not as well developed as amino terminal analysis.
- The method usually makes use of non-specific carboxypeptidases.
- Carboxypeptidases will sequentially hydrolyze polypeptides from the carboxy-terminus end. The released amino acid can be identified using liquid chromatographic methods, and the remaining polypeptide is available for further reactions.
- Various carboxypeptidases are available, usually they are not entirely non-specific (i.e. they have certain preferences):
Aromatics, aliphatics (hydrophobics)
Arginine, Lysine, Ornithine
Sometimes the choice of which carboxypeptidase to use is based upon the expected sequence information. In these types of experiments:
- samples are taken at different time points during the digestion
- free amino acids are separated from polypeptides
- the released amino acids are identified via amino acid analysis (liquid chromatography).
C-terminal analysis is usually only accurately for identification of the last half-dozen residues or so in a polypeptide.
One of the obvious problems with protein sequencing is that even if the N-terminal is not "blocked" only limited sequence infomation can be obtained from an intact polypeptide (i.e. only about two dozen from the N-terminal and half a dozen from the C-terminal).
How can sequence information for the entire polypeptide be obtained?
One method is that of peptide mapping. Peptide mapping makes use of proteolytic cleavages of the polypeptide to produce smaller polypeptides. These smaller polypeptides can then be isoloted from one another and subject to sequence analysis.
How do we order the different sequences which we obtain?
One of the easiest ways is to repeat the experiment, but with a protease with a different specificity, and in this way obtain overlapping sequence information.
Cleavage after Tyr, Phe and Trp; some cleavage after Leu, Met and Ala
Cleavage after Lys, Ala and Tyr
Cleavage after Arg, less after Lys
Cleavage after Glu, less after Asp
Figure 3.5.3: Overlapping cleavage products
Overlapping sequence information can allow you to align the peptides in the correct order and determine the sequence of the original large polypeptide (i.e. protein).
One problem which can arise deals with Cysteine residues and the nature of any covalent disulfide bridges in the protein.
- Any "peptide" mobilities (on either liquid chromatographic or PAGE analyses) which split into two smaller peptides after treatment with a reducing agent (such as b-ME) indicate the presence of a cysteine mediated disulfide bond.
- Upon sequencing these peptides should each contain a cysteine residue. If each peptide has only one cysteine then the disulfide bond assignment is unambiguous.
Figure 3.5.4: Cysteine residues in cleavage products
Corresponding genetic information
Once we have partial, or complete, peptide sequence information we can begin to identify and isolate the corresponding genetic information. This is the main goal. Once we have the corresponding genetic information it may be possible to produce relatively large amounts of the desired polypeptide.
Since we know the genetic code, we can back translate any polypeptide sequence into a corresponding genetic sequence.
- Thus, from the amino acid sequence we could synthesize an artificial gene which would code for the protein of interest.
- Since many amino acids are coded for by more than one codon, there is potential ambiguity with regard to the original exact genetic sequence.
Number of Codons
Phe, Tyr, His, Gln, Asn, Lys, Asp, Glu, Cys
Val, Pro, Thr, Ala, Gly
Leu, Arg, Ser
However, making sure we back translate in such a way as to faithfully duplicate the original genetic sequence may not be critical - a correct protein sequence is the overall goal.
In fact, if we are attempting to express the protein in another organism (say expressing a mammalian gene in a bacterial system) we may actually prefer to choose a codon bias appropriate for the expression host organism.
Synthetic genes for small proteins are a reasonable way to proceed; this is one way in which human insulin has been expressed in bacterial systems.
- However, automated synthesis of DNA oligonucleotides is practical for polymer lengths of approximately 60-90 bases or less (about 20-30 amino acids).
- Furthermore, the method of construction of synthetic genes typically calls for overlapping complementary oligonucleotides (to be ligated into a single duplex DNA gene "cassette").
Thus, many oligonucleotides are required for even a single small synthetic gene.
Figure 3.5.5: Synthetic gene construction
One way to improve upon the above method of synthetic gene construction is with a direct PCR approach. This method does not utilize ligase, or even oligonucleotides that butt together. Instead, with this method many (~100) different overlapping oligonucleotides are simultaneously used in a PCR reaction. Their sequence complementarity can be represented as follows:
The entire set of oligonucleotides may not line up to give the entire gene, but that is alright. We will do multiple rounds of PCR with the idea that some complementary oligo's will anneal and be extended and will lead, bit by bit, to construction of a contiguous synthetic gene:
On the next PCR cycle, some of these extended fragments will anneal with others:
These will be extended via the PCR and can go on to anneal with other larger PCR fragments. Eventually, the entire gene will be constructed. However, since the efficiency of construction of the full-length gene is probably not going to be very good, we need to conduct a subsequent PCR experiment to amplify the full-length gene (using outer primers). The principle features of this method are summarized as follows:
- Many (as many as 1-2 hundred) overlapping oligo's are combined in a single PCR reaction
- The oligos are designed to be as long as possible (~100mers) with limited overlap (~20 bases)
- The full-length gene is constructed in an initial (low yield) PCR experiment
- This full length gene is amplified with a subsequent typical PCR experiment using outer primers.
3.5: Protein Sequencing, Peptide Mapping, Synthetic Genes - Biology
BACKGROUND INFORMATION: You might want to consult Robert Russell's Guide to Structure Prediction. For the biochemical properties of amino acids see PROWL, Amino Acid Hydrophobicity and Amino Acid Chart and Reference Table (GenScript) . If you are specifically interested in antibodies I would recommend that you visit "The Antibody Resource Page."
Amino acid composition & Mass &ndash ProtParam (ExPASy, Switzerland)
Isoelectric Point - Compute pI/Mw tool (ExPASy, Switzerland). If you want a plot of the relationship between charge and pH use ProteinChemist (ProteinChemist.com) or JVirGel Proteomic Tools (PRODORIC Net, Germany).
Mass, pI, composition and mol% acidic, basic, aromatic, polar etc. amino acids - PEPSTATS (EMBOSS). Biochemistry-online (Vitalonic, Russia) gives one % composition, molecular weight, pI, and charge at any desired pH.
Peptide Molecular Weight Calculator (GenScript) - the online calculator determines the chemical formula and molecular weight of your peptide of interest. You can also specify post-translational modifications, such as N- and C- terminal modifications and positioning of disulfide bridges, to obtain more accurate outputs.
Isoelectric Point Calculator 2.0 (IPC 2.0) - is a server for the prediction of isoelectric points and pKa values using a mixture of deep learning and support vector regression models. The prediction accuracy (RMSD) of IPC 2.0 for proteins and peptides outperforms previous algorithms. ( Reference: Kozlowski LP (2021) Nucl. Acids Res. Web Server issue ).
Composition/Molecular Weight Calculation (Georgetown University Medical Center, U.S.A.) - the only problem with this site is that when run in batch mode it does not identify the sequence by name, merely sequential number
Protein calculator (C. Putnam, The Scripps Research Institute, U.S.A.) - calculates mass, pI, charge at a given pH, counts amino acid residues etc.
Tm Predictor (P.C. Lyu Lab., National Tsing-Hua University, Taiwan) - calculates the theoretical protein melting temperature.
Antigenicity and allergenicity: a good place to start would be The Immune Epitope Database (IEDB)
Abie Pro Peptide Antibody Design (Chang Bioscience)
Allergenicity servers: AllerTOP ( Reference : Dimitrov, I. et al. 2013. BMC Bioinformatics 14(Suppl 6): S4), AlgPred - prediction of allergenic proteins and mapping of IgE epitopes ( Reference: Saha, S. and Raghava, G.P.S. 2006. Nucleic Acids Research 34: W202-W209.), and SDAP - Structural Database of Allergenic Proteins ( Reference: Ivanciuc, O. et al. 2003. Nucleic Acids Res. 31: 359-362).
EpiToolKit - is a virtual workbench for immunological questions with a focus on vaccine design. It offers an array of immunoinformatics tools covering MHC genotyping, epitope and neo-epitope prediction, epitope selection for vaccine design, and epitope assembly. In its recently re-implemented version 2.0, EpiToolKit provides a range of new functionality and for the first time allows combining tools into complex workflows. For inexperienced users it offers simplified interfaces to guide the users through the analysis of complex immunological data sets. ( Reference: Schubert S et al. (2015) Bioinformatics 31(13): 2211&ndash2213).
VIOLIN - Vaccine Investigation and OnLine Information Network - allows easy curation, comparison and analysis of vaccine-related research data across various human pathogens VIOLIN is expected to become a centralized source of vaccine information and to provide investigators in basic and clinical sciences with curated data and bioinformatics tools for vaccine research and development. VBLAST: Customized BLAST Search for Vaccine Research allows various search strategies against against 77 genomes of 34 pathogens. ( Reference: He, Y. et al. 2014. Nucleic Acids Res. 42(Database issue): D1124-32).
SVMTriP - is a new method to predict antigenic epitope with lastest sequence input from IEDB database. In our method, Support Vector Machine (SVM) has been utilized by combining the Tri-peptide similarity and Propensity scores (SVMTriP) in order to achieve the better prediction performance. Moreover, SVMTriP is capable of recognizing viral peptides from a human protein sequence background. ( Reference: Yao B et al. (2012) PLoS One 7(9): e45152).
Solubility and crystalizability:
EnzymeMiner - offers automated mining of soluble enzymes with diverse structures, catalytic properties and stabilities. The solubility prediction employs the in-house SoluProt predictor developed using machine learning.( Reference: Hon J et al. 2020. Nucl Acids Res 48 (W1): W104&ndashW109).
ESPRESSO (EStimation of PRotein ExpreSsion and SOlubility) - is a sequence-based predictor for estimating protein expression and solubility for three different protein expression systems: in vivo Escherichia coli, Brevibacillus, and wheat germ cell-free. ( Reference: Hirose S, & Noguchi T. 2013. Proteomics. 13:1444-1456).
SABLE - Accurate sequence-based prediction of relative Solvent AccessiBiLitiEs,secondary structures and transmembrane domains for proteins of unknown structure. ( Reference: Adamczak R et al. 2004. Proteins 56:753-767).
SPpred (Soluble Protein prediction) (Bioinformatics Center, Institute of Microbial Technology, Chandigarh, India) - is a web-server for predicting solubility of a protein on over expression in E.coli. The prediction is done by hybrid of SVM model trained on PSSM profile generated by PSI-BLAST search of 'nr' protein database and splitted amino acid composition.
Protein&ndashSol - is a web server for predicting protein solubility. Using available data for Escherichia coli protein solubility in a cell-free expression system, 35 sequence-based properties are calculated. Feature weights are determined from separation of low and high solubility subsets. The model returns a predicted solubility and an indication of the features which deviate most from average values. ( Reference: Hebditch M et al. 2017. Bioinformatics 33(19): 3098&ndash3100).
CamSol - for the rational design of protein variants with enhanced solubility. The method works by performing a rapid computational screening of tens of thousand of mutations to identify those with the greatest impact on the solubility of the target protein while maintaining its native state and biological activity. ( Reference: Sormanni P et al. (2015) J Molec Biol 427(2): 478-490). N.B. Requires registration.
Surface Entropy Reduction p rediction (SERp) - this exploratory tool aims to aid identification of sites that are most suitable for mutation designed to enhance crystallizability by a Surface Entropy Reduction approach. ( Reference: Goldschmidt L. et al. 2007. Protein Science. 16:1569-1576)
CRYSTALP2 - for in-silico prediction of protein crystallization propensity. ( Reference: Kurgan L, et al. 2009. BMC Structural Biology 9: 50) and, PPCpred - sequence-based prediction of propensity for production of diffraction-quality crystals, production of crystals, purification and production of the protein material.( Reference: M.J. Mizianty & L. Kurgan. 2011. Bioinformatics 27: i24-i33).
Antimicrobial peptides, vaccines and toxins:
APD (Antimicrobial Peptide Database) ( Reference: Wang, Z. and Wang, G 2004. Nucl. Acids Res.32: D590-D592)
The Type III Secretion System (T3SS) is an essential mechanism for host-pathogen interaction in the infection process. The proteins secreted through the T3SSmachinery of many Gram-negative bacteria are known as T3SS effectors (T3SEs). These can either be localized subcellularly in the host, or be part of the needle tip of the T3SS that interacts directly with the host membrane to bring other effectors into the target cell. T3SEdb represents such an effort to assemble a comprehensive database of all experimentally determined and putative T3SEs into a web-accessible site. BLAST search is available. ( Reference: Tay DM et al. 2010. BMC Bioinformatics. 11 Suppl 7:S4).
Effective (University of Vienna, Austria & Technical University of Munich, Germany) - Bacterial protein secretion is the key virulence mechanism of symbiotic and pathogenic bacteria. Thereby effector proteins are transported from the bacterial cytosol into the extracellular medium or directly into the eukaryotic host cell. The Effective portal provides precalculated predictions on bacterial effectors in all publicly available pathogenic and symbiontic genomes as well as the possibility for the user to predict effectors in own protein sequence data.
Vaxign is the first web-based vaccine design system that predicts vaccine targets based on genome sequences using the strategy of reverse vaccinology. Predicted features in the Vaxign pipeline include protein subcellular location, transmembrane helices, adhesin probability, conservation to human and/or mouse proteins, sequence exclusion from genome(s) of nonpathogenic strain(s), and epitope binding to MHC class I and class II. The precomputed Vaxign database contains prediction of vaccine targets for >350 genomes. ( Reference: He Y et al. 2010. J Biomed Biotechnol. 2010: 297505). A newer version Vaxign 2 Beta is available here.
VacTarBac is a platform which stores vaccine candidate against several pathogenic bacteria. The vaccine are designed on the basis of their probabilty to act as epitope, thus have the potential to induce any of the several arm of immune system. These epitopes have been predicted against the virulence factor and essentail genes of 14 bacterial species. ( Reference: Nagpal G et al. (2018) Front Immunol. 9: 2280).
Abpred - will take a single amino acid sequence for a Fv and calculate the predicted performance on 12 biophysical platforms ( Reference: Hebditch M & J Warwicker (2019) PeerJ. 7: e8199).
T3SE - Type III secretion system effector prediction ( Reference: Löwer M, & Schneider G. 2009. PLoS One. 4:e5917. Erratum in: PLoS One. 20094(7).
SIEVE Server is a public web tool for prediction of type III secreted effectors. The SIEVE Server scores potential secreted effectors from genomes of bacterial pathogens with type III secretion systems using a model learned from known secreted proteins. The SIEVE Server requires only protein sequences of proteins to be screened and returns a conservative probability that each input protein is a type III secreted effector. ( Reference: McDermott JE et al. 2011. Infect Immun. 79:23-32).
Circular Dichroism (Birkbeck College, School of Crystalography, England) DICHROWEB is an interactive web site which allows the deconvolution of data from Circular Dichroism spectroscopy experiments. It offers an interface to a range of deconvolution algorithms (CONTINLL, SELCON3, CDSSTR, VARSLC, K2D).
K2D2: Prediction of percentages of protein secondary structure from CD spectra - allows analysis of 41 CD spectrum data points ranging from 200 nm to 240 nm or or 51 data points for the 190-240 nm range ( Reference: Perez-Iratxeta C & Andrade-Navarro MA. 2008. BMC Structural Biology 2008, 8:25)
K2D3 is a web server to estimate the a helix and ß strand content of a protein from its circular dichroism spectrum. K2D3 uses a database of theoretical spectra derived with Dichrocalc ( Reference: Louis-Jeune C et al. 2012. Proteins: Structure, Function, & Bioinformatics 80: 374&ndash381)
DiANNA - will predict cysteine oxidation state (76% accuracy), cysteine pairs (81% accuracy) and disulfide bond connectivity (86% accuracy). ( Reference: Nucl. Acids Res. 33: W230-W232).
CYSREDOX (Rockefeller University, U.S.A.) and CYSPRED (CIRB Biocomputing Group, University of Bologna, Italy) calculate the redox state of cysteine residues in proteins.
Hydrophobicity Plotter ( Innovagen ) - and Protein Hydroplotter - sellect under Tools (ProteinLounge, San Diego, CA ).
Proteolysis and Mass Spectrometry:
Proteolysis - PeptideCutter (ExPASy, Switzerland) which also predicts cleavage sites for enzymes and chemicals. An alternative proteolysis site is Mobility_plot 4.1 (Advanced Proteolytic Fingerprinting, IGH, France).
For more sophisticated protein analysis involving mass spectroscopy ExPasy has introduced FindMod to predict potential protein post-translational modifications in peptides and, GlycoMod which can predict the possible oligosaccharide structures that occur on proteins from their experimentally determined masses.
ProFound - is a tool for searching a protein sequence database using information from mass spectra of peptide maps. A Bayesian algorithm is used to rank the protein sequences in the database according to their probability of producing the peptide map. A simplified version can be accessed here (Rockefeller University, New York, U.S.A.) . One cannot use one's own protein database.
ProteinProspector (University of California) - offers a wide variety of tools (e.g. MS-Fit, MS-Tag, MS-Seq, MS-Pattern, MS-Homology) for the protein mass spectroscopist.
Repeats in protein sequences can be discovered using Radar ( R apid A utomatic D etection and A lignment of R epeats, European Bioinformatics Institute) or REPRO ( Reference: George RA. & Heringa J. 2000. Trends Biochem. Sci. 25: 515-517).
REPPER (REPeats and their PERiodicities) - detects and analyzes regions with short gapless repeats in proteins. It finds periodicities by Fourier Transform (FTwin) and internal similarity analysis (REPwin). FTwin assigns numerical values to amino acids that reflect certain properties, for instance hydrophobicity, and gives information on corresponding periodicities. REPwin uses self-alignments and displays repeats that reveal significant internal similarities. They are complemented by PSIPRED and coiled coil prediction (COILS), making the server a useful analytical tool for fibrous proteins. ( Reference: M. Gruber et al. 2005. Nucl. Acids Res. 33: W239-W243).
JVirGel calculation of virtual two-dimensional protein gels - creates virtual 2D proteomes from a huge list of eukaryotes & prokaryotes (or an individual protein). Two versions: html (limited) and Java applet (incredible but you need to install Java Runtime Environment. ( Reference: K. Hiller et al. 2003. Nucl. Acids Res. 31: 3862-3865).
Draw Virtual Two-Dimensional Protein Gels (PRODORIC Net, Germany) - using your own protein sequence data or for different organisms.
Scratch Protein Predictor - (Institute for Genomics and Bioinformatics, University California, Irvine) - programs include: ACCpro: the relative solvent accessibility of protein residues CMAPpro: Prediction of amino acid contact maps COBEpro: Prediction of continuous B-cell epitopes CONpro: predicts whether the number of contacts of each residue in a protein is above or below the average for that residue DIpro: Prediction of disulphide bridges DISpro: Prediction of disordered regions DOMpro: Prediction of domains SSpro: Prediction of protein secondary structure SVMcon: Prediction of amino acid contact maps using Support Vector Machines and, 3Dpro: Prediction of protein tertiary structure (Ab Initio).
Gene Mutagenesis Designer (GenScript) is developed to make your design of point DNA mutagenesis straightforward to facilitate gene mutation. To perform DNA mutagenesis from wild type, simply input your starting sequence of wild type gene into the field below, and then click on the &ldquofrom selection&rdquo button to select the amino acid(s) of interest. Consequently, the new gene sequence encoding mutated protein will be generated upon a click &ldquosubmit&rdquo. You can select a number of expression systems.
I-Mutant2.0: predictor of protein stability changes upon mutation - choose either a PDB reference number or paste your own protein. The answer (by email) indicates whether the protein is more or less stable, a fact which could be of use in designing "better" proteins. ( Reference: E. Capriotti et al. 2005. Nucl. Acids Res. 33: W306-W310).
SIFT - The Sorting Intolerant from Tolerant (SIFT) algorithm predicts the effect of coding variants on protein function i.e. it predicts whether an amino acid substitution affects protein function based on sequence homology and the physical properties of amino acids. SIFT can be applied to naturally occurring nonsynonymous polymorphisms and laboratory-induced missense mutations. ( Reference: N-L Sim et al. 2012. Nucleic Acids Research 40(1): W452&ndashW457).
mCSM-membrane - predicts the effects of mutations on transmembrane proteins. ( Reference: Pires DEV et al. 2020. Nucl Acids Res 48 (W1): W147&ndashW153).
3.5: Protein Sequencing, Peptide Mapping, Synthetic Genes - Biology
The cDNA cloning and expression in vitro and in eukaryotic cells of a novel protein isolated from human articular cartilage, cartilage intermediate layer protein (CILP) is described. A single 4.2-kilobase mRNA detected in human articular cartilage encodes a polypeptide of 1184 amino acids with a calculated molecular mass of 132.5 kDa. The protein has a putative signal peptide of 21 amino acids, and is a proform of two polypeptides. The amino-terminal half corresponds to CILP (molecular mass of 78.5 kDa, not including post-translational modifications) and the carboxyl-terminal half corresponds to a protein homologous to a porcine nucleotide pyrophosphohydrolase, NTPPHase (molecular mass of 51.8 kDa, not including post-translational modifications). CILP has 30 cysteines and six putative N-glycosylation sites. The human homolog of porcine NTPPHase described here contains 10 cysteine residues and two putative N-glycosylation sites. In the precursor protein the NTPPHase region is immediately preceded by a tetrapeptide conforming to a furin proteinase cleavage consensus sequence. Expression of the full-length cDNA in a cell-free translation system and in COS-7 or EBNA cells indicates that the precursor protein is synthesized as a single polypeptide chain that is processed, possibly by a furin-like protease, into two polypeptides upon or preceding secretion.
This work was supported by the Swedish Medical Research Council, Konung Gustaf V's 80-årsfond, Greta och Johan Kock's stiftelser, Axel och Margaret Ax:son Johnsons stiftelse, AlfredÖsterlund's Stiftelse and the Medical Faculty, Lund University.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) AF035408.
3.5: Protein Sequencing, Peptide Mapping, Synthetic Genes - Biology
Advanced laboratories and facilities are essential for the college's pioneering research programs. Explore the college's core facilities below.
There are a number of facilities that support University of Texas at Austin researchers in chemistry and related areas.
Biomedical research facilities
There are a number of facilities that support cellular and molecular biology research and computational biology at The University of Texas at Austin. Mostly housed within the College's Center for Biomedical Research Support, these facilities offer a full range of services in nucleic acid and protein sequencing, peptide synthesis, mass spectrometry, protein purification and analysis, DNA microarrays, x-ray chrystallography, and transgenic - knockout mice. These facilities include:
The NHB Research Greenhouse, located on the roof of the Norman Hackerman Building (NHB), provides large greenhouse rooms as well as walk-in and reach-in growth chambers for plant researchers across the university.
The Biomedical Imaging Center (BIC) is home to a high-field (3 Tesla) MRIt, an image analysis computer suite, test rooms, fully outfitted electronics & machine shops, offices, and a conference/classroom area.
Nanotechnology Core Facilities
The Texas Materials Institute is the home for what was previously the Center for Nano and Molecular Science and Technology. Facilities include scanning probe microscopy, nano device fabrication and testing, and electronic and vibrational spectroscopy.
- Nano Fabrication and Characterization Facility
- Microelectronic Research Facility
- X-ray Analysis Facility
- Surface Analysis Facility
- Electron and Scanning Probe Microscopy
- Polymer Characterization Facility
The Physics Machine Shop, located in RL Moore, designs and fabricates specialized research equipment and instruments for research labs across the campus. The Physics Machine Shop also manages a Cryogenics Shop.
Peptide Mapping by LC-MS/MS Services
A setup fee may apply depending on number of samples and sample preparation time. The above quoted prices are based on analysis using our standardized methods, proven to work for the majority of antibodies and proteins. Method development options may be necessary for atypical proteins with unique properties.
Price/availability/specifications subject to change without notice. Unless otherwise indicated, our catalog and customized products are for research use only and not intended for human or animal diagnostic or therapeutic use.
Or leave a message with a formal purchase
Using peptide mapping, subtle changes to the primary structure of a protein or antibody can be identified that may affect the activity of the protein or the binding affinity. Peptide mapping by LC-MS/MS is one of the most powerful qualitative assays to confirm the primary sequence of proteins or antibodies. This service can be used to compare different lots of the same proteins, antibodies or biosimilars.
Service of peptide mapping analysis by LC-MS/MS includes:
A. The antibody/protein of interest is denatured, reduced, and digested with a proteolytic enzyme such as LysC or trypsin.
B. Optional: a second proteolytic enzyme is used to increase the sequence coverage of the protein.
C. The resulting peptide mixtures are separated on a reversed-phase HPLC. The chromatograph of UV absorbance is generated at UV 214 nm.
D. After HPLC separation, further analysis is performed by a high resolution mass spectrometry (Thermo Fisher Orbitrap Velos or Fusion).
E. A peptide map of the protein is generated for comparison with a reference protein.
F. The peaks are analyzed to the primary sequence.
G. Optional (two or more samples): a comparability evaluation is performed to assess lot-to-lot variation between proteins or to compare biosimilars.
- Confirmation of primary sequences (sequence coverage: 60-90% using one enzyme > 98% using two or more enzymes).
- Glycoprofiling of N-linked oligosaccharides (heterogeneity of glycoforms & approximation of site occupancy).
- Identification & approx quantification of PTMs such as deamidation, oxidation, N-term pyroglutamate formation and C-term Lys processing (modified amino acid position and % modification can be detected).
- Similarity assessment between different lots.
- Comparability assessment for biosimilars.
Sample treatment: Denaturation, reduction/alkylation and enzymatic digestion followed by LC-MS or LC-MS/MS.
3.5: Protein Sequencing, Peptide Mapping, Synthetic Genes - Biology
Molecular Biology Services
Sangon Biotech have developed particular technology for obtain DNA fragments ("gene") with specific functions either by cloning from natural existing organism or by chemical gene synthesis. Cloning is usually more convenient than gene synthesis, but cloning is also time-consuming and sometimes can be difficult. Sangon Biotech not only help you to obtain the desired gene, but also help you to subcloning the desired gene into any vectors to save your time and money.
- Direct gene cloning with known sequence from known source
- Norvel gene cloning using RACE (Rapid Amplification of cDNA Ends) technology
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product (either proteins or non-protein coding genes such as miRNA). The regulation of gene expression process was strictly controlled in a complicated network in vivo. Gene expression profiling indicates the role of a particular gene and the changes in gene expression is associated with many biological process such as disease developing. So monitor the changes in gene expression becoming an more and more important method to monitor the "action" of particular genes.
Real-time PCR analysis is the gold standard for quantifying gene expression. We have developed methods for sensitive, accurate quantification of mRNA or microRNA using real-time PCR. Both TaqMan probe-based analysis and SYBR Green dye-based analysis are available. Individual samples or 96 or 384 well plates can be analyzed.
- mRNA Real Time Fluorescent Quantitative RT-PCR
- microRNA Real Time Fluorescent Quantitative RT-PCR
Microsatellite (STR) Genotyping
Microsatellites, also known as short tandem repeats (STR), are widely used molecular markers in genetics. Sangon Biotech offers a complete microsatellite (MS) genotyping service.
Single Nucleotide Polymorphism (SNP) Genotying
SNPs are one of the most common types of genetic variation which is associated with many diseases. SNP genotyping is the measurement of genetic variations of single nucleotide polymorphisms (SNPs) between members of a species. Sangon Biotech have developed several SNP genotyping platforms for different requirement including:
- Direct Sequencing
- Restricted Fragment Length Polymorphisms (RFLP)
- TaqMan Probe
- Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS)
cDNA library construction is a powful tool for gene expression, gene function research, norvel gene characterization, and many other applications. But obtain high-quality cDNA libraries is time-consuming and can be very challenging. Sangon Biotech has over 10 years of experience in generating cDNA libraries from various of samples. Thanks to our comprehensive sequencing platform. cDNA libraries with ESTs (expressed sequence tag) can also be sequenced and analysized here.
- Identification of Bacteria or Fungi
- PCR-DGGE (Denaturing Gradient Gel Electrophoresis) analysis for the identification of microbial populations of Bacteria
- Microbial Population Analyzing
SNP service content price and procedure
SNP testing service platform
- Direct sequencing
- Restricted fragment length polymorphisms (RLFP)
- TaqMan Probe
- MALDI-TOF MS
- Illumina beadchip SNP genotyping
- Illumina beadchip methylation
- Illumina beadchip gene expression profiles
- Illumina beadchip microRNA expression profiles
|Services||Price ($)||Turnaround Time||Remarks|
|Known gene cloning from Prokaryotic or eukaryotic genome||Minimum charge at $80 ( |
Bisulfite modification and sequencing method (BSP)
Frommer et al (1992) proposed methods of DNA methylation analysis, this method is reliable and precise. procedures:
Methylation specific PCR method (Methylation-Specific PCR, MS-PCR)
Herman et al (1996) in the use of bisulfite treatment on the basis of the new methods. High sensitivity of this method is mainly used for qualitative research. Procedures:
Lab report (experimental steps, including the BSP method statistical results of 5 clones), PCR electrophoresis photographs, sequencing trace files.
10.1 Adenovirus packaging services
10.2 Lentiviral packaging services
11.RNA Interference Service
Reports include: siRNA sequences, construct vectors sequencing report, quantitative PCR test report, Western Blot image and all the experimental data and detailed experimental procedures
3.5: Protein Sequencing, Peptide Mapping, Synthetic Genes - Biology
One of the Largest Professionals in Oligo Synthesis
Oligonucleotide synthesis is the chemical synthesis of short nucleic acids chains with desired sequence . The capability of "create" nucleic acids from building blocks (2'-deoxynucleosides, ribonucleosides, or chemically modified nucleosides, e.g. Florescence labeled deoxynucleosides.) has speeded up research in molecular biology. Oligonucleotides are widely used in most laboratory and it is extremely useful in various applications in molecular biology and medicine.
The development of oligonucleotide synthesis technology started in the early 1950s. The most widely used technique for oligonucleotide synthesis currently is the solid-phase synthesis using phosphoramidite modified nucleosides as primary elements. The synthesis of DNA and RNA is carried our in 3'-5'direction, on the opposite of the natural 5'-3' direction. The synthesis process has been used since the late 1970s and become more and more automated during the past 30 years. Now the major steps of oligonucleotide synthesis are performed by automated synthesizers. The limits for the length of oligonucleotide being synthesized and the purity of oligonucleotides also increases.
As the world's leading supplier of custom oligonucleotides, Sangon Biotech have over 15 years of experience perfecting the process of making oligos. The process of oligonucleotide synthesis in Sangon Biotech was fully automated and integrated and now we have a capacity of 10,000 oligos per day, with a length limit up to 130-bp and 3 kinds of purification methods (HAP, PAGE and HPLC). We also able to successfully synthesize difficult and unusual oligonucleotides. A complicated quality assurance system was established to ensure the process was carried under strict control.
Frequently Asked Questions and Answers
Q: What's the storage condition of oligonucleotide ?
Synthesized oligonucleotides are provided in lyophilized powder. Oligonucleotides in the form of lyophilized powder are relatively stable, it can be transported at ambient temperature and stored at room temperature for a couple of days. Oligos in lyophilized powder is stable for years when stored at -20℃. After reception, oligonucleotides are recommended to be dissolved in TE buffer to 100 ?M and stored at -20℃. It is stable for several months and freeze-thraw cycles should be avoided. A work solution in 10 ?M is always stable for several days if stored at 2-8℃.
Q: How do I measure the amount of oligonucleotides ?
An OD reading is a quick way of estimating how much DNA is contained in the solution, by measuring the absorbance at 260 nm. 1 OD is approximately 33 ?g of crude oligo DNA. It should be noted that, for especially desalted oligos, the OD reading is a measurement of the total amount of nucleic acid which includes both the full-length and failed sequences.
Q: What is HAP purification and how do I choose it ?
High Affinity Purification (HAP) is a patented, novel purification method for custom oligos developed by Sangon Biotech. DMT-ON-Oligo in the crude oligo mixture is first selectively absorbed on a high affinity resin in HAP column while incomplete oligos pass through. Final products is obtained by removing the protection group of DMT under mild acidic conditions. HAP method provides two major advantages, high purity superior to De-Salted method (Purity of a standard 20 bases-HAP is >85%, 30 bases-HAP > 80% etc. ), and low cost compare to PAGE or HPLC methods. Oligos produced by the HAP method has such high purity that they can be directly used for any downstream experiments such as PCR, DNA Sequencing, Gene Synthesis, and Mutagenesis. At present, the most economic method to produce oligos is the De-Salted method, which however, yields products poor in purity. For example, the purity of 20-mer, 40-mer and 60-mer is approximately 68%, 45% and 30% respectively. This is calculated based on the 4 steps in DNA synthesis: De-DMT, Coupling, Oxidation and Capping. The average yield of each cycle is about 98%. The purity of 20-mer is therefore (0.98)20-1 = 68% of 40-mer = (0.98)40-1 = 45% and of 60-mer = (0.98)60-1 = 30%. Most laboratories use De-Salted oligos despite of their inferior purity because higher quality oligos produced by alternative methods such as PAGE, HPLC, and OPC are too costly. HAP presents the perfect alternative for high purity oligos at lowest prices. In fact, price is even lower than those of De-Salted method in some cases.
Q: What are the various purification options ?
During the synthesis of oligonucleotides, the crude products contain undesired oligos caused by side reaction as well as full-length oligos. Purification procedures are required to remove these undesired products and/or other component such as salts. Sangon Biotech offer 3 kinds of purification options for oligonucleotides.
Reverse Phase HPLC: This method also uses the 5' trityl protecting group as a means of binding to the column. This method works well for oligos up to 55 bases and generally 90 to 95% pure.
Protein identification in the post-genome era: the rapid rise of proteomics
Most advances in biology can usually be traced back to the development of a new technique: the recent explosion in sequence information in the databases arose from the pioneering work on separation methods by Frederick Sanger which paved the way for the development of protein (Sanger, 1945) and DNA/RNA (Maxam & Gilbert, 1977 Sanger, 1981) sequencing and culminated in the receipt of two Nobel prizes by Sanger. The initial phase of sequence database expansion was slow due to the tedious and slow nature of protein sequencing. Peptide sequencing was carried out manually and the complete analysis of a protein was tiresome, requiring the isolation of sufficient peptides from several digests of the target protein using proteases of different specialities to collect an overlapping set of fragments which cover the whole sequence. Protein sequencing gained momentum when the phenylisothiocyanate sequencing chemistry developed by Edman in 1949 was automated (Edman & Begg, 1967) and a commercial instrument requiring lower amounts (nanomoles) of sample was put on the market. Further technical advances such as novel valves to deal with small volumes of aggressive chemicals, the introduction of high pressure liquid chromatography (HPLC), and novel supports for sample immobilization, were all combined in the first gas phase sequencers, greatly increasing the sensitivity and allowing automated data collection (Hewick et al . 1981) and analysis. The new instruments with a sensitivity in the low picomole range appeared as rapid advances in DNA technology such as the development of restriction mapping (Danna et al . 1973), cloning (Cohen et al . 1973) and the dideoxynucleotide sequencing chemistry were threatening to make protein chemistry a relic of the past (Malcolm, 1978).
Manual sequence workflows
Using the tools within the Bio Tool Kit, users can “walk” up or down an MS/MS spectrum to determine the sequence of a peptide (Figure 3). This process is straightforward and is accomplished by following these steps:
Peptide sequences can also be matched to MS/MS data with the peptide fragment pane. Here, a table of theoretical fragment ions from the proposed sequence is matched to ions found in the data. All matches are highlighted within the table and labeled in the MS/MS spectrum. The tool considers multiple different fragment ion types and will also find and highlight modifications (Figure 4).
Figure 3. Manual peptide sequencing. The peak with nominal mass 1092 was highlighted as the starting point for sequencing the peptide from the MS/MS data. The next peaks were then selected for consideration, one after another, with the final sequence shown above the spectrum.× Close
Figure 4: Peptide fragment pane. The theoretical fragment ions from the proposed sequence are matched to the fragment ions found in the MS/MS data. All matches are highlighted in the table and annotated in the spectrum.× Close
3.5: Protein Sequencing, Peptide Mapping, Synthetic Genes - Biology
Below is a list of core facilities and advanced laboratories in the college.
DEPARTMENT OF CHEMISTRY
Interdisciplinary Life Sciences Graduate Programs
The ILS core facilities support cellular and molecular biology research at The University of Texas at Austin. The facilities offer a full range of services in nucleic acid and protein sequencing, peptide synthesis, mass spectrometry, protein purification and analysis, DNA microarrays, x-ray chrystallography, and transgenic - knockout mice. The ICMB core facilities include:
IMAGING RESEARCH CENTER
The Imaging Research Center is home to a high-field (3 Tesla) MRIt, an image analysis computer suite, test rooms, fully outfitted electronics & machine shops, offices, and a conference/classroom area.
NANOTECHNOLOGY CORE FACILITIES
The Center for Nano and Molecular Science and Technology facilities located in the FNT building features a variety of facilities that support state-of-the-art teaching activities and high-level scientific research.
The Department of Physics provides a number of facilities and services for use by faculty, staff, and students within the Department. Many facilities are also available for use by other university-affiliated students and staff.
PLANT RESOURCES CENTER
The Plant Resources Center (TEX-LL) with over 1,000,000 specimens is the largest herbarium in the southwestern United States and ranks fifth among U.S. university herbaria and twelfth across the nation. TEX-LL, with about a quarter of its specimens from Texas, has the largest holdings of Texas plants in the world. Nearly one half of the specimens at TEX-LL are from Latin America, with an especially strong representation of Mexico and northern Central America. Presently the number of vascular plant collections inserted in the herbarium is growing at an approximate rate of 16,400 specimens per year.
TEXAS PETAWATT LASER
The college is currently home to the highest power laser in the world, the Texas Petawatt Laser, which, when turned on, has the power output of more than 2,000 times the output of all power plants in the United States. (A petawatt is one quadrillion watts.) The laser is brighter than sunlight on the surface of the sun, but it only lasts for an instant, a 10th of a trillionth of a second (0.0000000000001 second).
TEXAS NATURAL SCIENCE CENTER
With many facilities at the J.J. Pickle research campus in North Austin, the Texas Natural Science Center is home to some of the most extensive collections of invertebrate and vertebrate fossils and natural history collections in the country. A high-resolution X-ray CT (Computed Tomography) scanner is available at the Vertebrate Paleontology Lab.
UTEX CULTURE COLLECTION OF ALGAE
The Culture Collection includes approximately 3,000 different strains of living algae, representing most major algal taxa. The primary function of UTEX is to provide algal cultures at modest cost to a user community. Cultures in the Collection are used for research, teaching, biotechnology development, and various other projects throughout the world.
Watch the video: What is a Chromosome? (January 2023).