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What is the fastest way to build an alanine scanning library?

What is the fastest way to build an alanine scanning library?



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For interfacial studies, I would like to build an alanine scanning library for one of my proteins examining 20 sites. I will ultimately express the gene using E.coli cell-free protein synthesis. I already have the template gene which was originally built using PCR assembly. Now, what's the best way to build a scanning library?


The fastest will change as time passes and better technologies are developed.

I think the fastest method existing at the moment is Shotgun Mutagenesis (provided by Integral Molecular Inc).

This does not employ any new method of doing that. They just provide a set of plasmids, that has all the possible mutations. The set itself is generated by automated DNA synthesis.

So if you don't have a DNA synthesizer with you then simply order the kit from the company.


Frontiers in Chemistry

The editor and reviewers' affiliations are the latest provided on their Loop research profiles and may not reflect their situation at the time of review.


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    2 Materials and methods

    The web application presents a simple user interface ( Fig. 1) that wraps functionality from the BudeAlaScan command-line application, which is described and benchmarked in detail elsewhere ( Ibarra et al., 2019). Briefly, the BudeAlaScan command-line application uses ISAMBARD ( Wood et al., 2017) to prepare input files for a custom version of the Bristol University Docking Engine (BUDE) ( McIntosh-Smith et al., 2012), which performs CASM using an empirical free-energy forcefield to estimate the free-energy of binding. The accuracy of BudeAlaScan has been experimentally benchmarked and compares favourably with other methods ( Ibarra et al., 2019), but is significantly faster than alternatives, making it ideal for use in an interactive web application.

    Overview of the BAlaS web application. (A) The main interface contains a structure visualizer and a panel for submitting jobs and displaying results. The scan panel is visible on the right and displays the results of a CASM job, with a table describing the energetic contribution of each residue. (B) The constellation tab displays energies calculated for each constellation, in this case from a residues mode job. (C) The jobs panel displays all jobs that have been submitted by the user. The user can display the results, create a link to the job that they can share, download the full output, or delete the job.

    Overview of the BAlaS web application. (A) The main interface contains a structure visualizer and a panel for submitting jobs and displaying results. The scan panel is visible on the right and displays the results of a CASM job, with a table describing the energetic contribution of each residue. (B) The constellation tab displays energies calculated for each constellation, in this case from a residues mode job. (C) The jobs panel displays all jobs that have been submitted by the user. The user can display the results, create a link to the job that they can share, download the full output, or delete the job.

    The front end of the BAlaS application is written in Elm (https://elm-lang.org/) and handles input validation, job management and results visualization. Structures are displayed using the PV JavaScript library ( Biasini, 2015). The backend architecture is relatively straightforward: NGINX is used to serve the application and the result files a NoSQL database (MongoDB) is used for the job queue and for storing results a simple script polls the database and runs BudeAlaScan jobs and a RESTful API, written using the Flask Python library (http://flask.pocoo.org/), is used by the front end to submit jobs and retrieve results. The whole application stack runs inside three Docker containers (https://www.docker.com/), which enables users to run the web application locally if desired.

    The constellation mode allows the user to combine multiple alanine mutations to find regions of the ‘ligand’ that interact cooperatively with the ‘receptor’, which we call hot constellations i.e. the ΔΔG when all residues are simultaneously mutated to alanine, is greater than the sum of the ΔΔG values when each residue is mutated to alanine individually. This is useful for highlighting regions of the ‘ligand’ that are key to forming the interaction with the ‘receptor’. Constellation jobs can be run in one of three modes: manual, residues and auto. In manual mode, the user specifies all the residues in a constellation. In residues mode, the user selects a list of residues and a constellation size, and all permutations of the residues equal to the size of the constellation are evaluated. In auto mode, the user defines a constellation size, a ΔΔG cut-off (kJ/mol) and a Cα-Cα distance cut-off (Å). Permutations of all residues with a ΔΔG above the cut-off are created for the defined constellation size, provided that the residues are within the Cα–Cα distance cut-off of each other.

    Finally, the jobs tab is used to manage the user’s jobs as well as retrieve and share results. Full results from the command-line application can be downloaded as a zip file and contains the ΔΔG data in text formats, as well as scripts for displaying the results in the molecular graphics programme Chimera. Users can plot the graphical data (json files) contained in the output easily through the replotAlaScan.py script in the BudeAlaScan package.


    References

    Littlewood, T. & Evan, G. I. Helix–Loop–Helix Transcription Factors (Oxford Univ. Press, New York, 1998).

    Winston, R. L. & Gottesfeld, J. M. Chem. Biol. 7, 245–251 (2000).

    Murre, C., McCaw, P. S. & Baltimore, D. Cell 56, 777– 783 (1989).

    Ferre-D'Amare, A. R., Prendergast, G. C., Ziff, E. B. & Burley, S. K. Nature 363, 38–45 ( 1993).

    Ma, P. C., Rould, M. A., Weintraub, H. & Pabo, C. O. Cell 77, 451–459 ( 1994).

    Grandori, C. & Eisenman, R. N. Trends Biochem. Sci. 22, 177–181 (1997).

    Grandori, C., Mac, J., Siebelt, F., Ayer, D. E. & Eisenman, R. N. EMBO J. 15, 4344– 4357 (1996).


    Materials and Methods

    Strain construction

    The original actin alanine scan allele strains were constructed with both a linked HIS3 marker and a linked tub2-201 allele in the β-tubulin gene that confers resistance to benomyl (Wertman and Drubin 1992). We were concerned that the β-tubulin mutation would contribute to the genetic interactions in our screens. Furthermore, in our CHI procedure, we prefer the mutant alleles be marked with a linked nourseothricin resistance (NAT r ) gene because this gives a very tight selection. Therefore, we undertook reconstructing the actin alanine scan alleles in a more suitable background. During this process, we discovered that seven of the original actin alanine scan mutants had additional mutations. We corrected all seven but found that two mutants previously reported to be recessive lethal alleles were in fact likely dominant lethal alleles and therefore could not be used in our analysis. The correction of these seven alleles and their phenotypic analysis is described in a letter to Genetics (Viggiano et al. 2010). This left 31 alanine scan alleles, marked with NAT r and without the β-tubulin mutation for CHI analysis. In addition, we included the act1-159 allele that encodes a filament-stabilizing mutant of actin (Belmont and Drubin 1998). Table 1 lists these alleles, their phenotypes, locations, and number of interactions.

    Table 1

    AlleleMutationPhenotypeLocationCHI Interactions
    act1-101D363A,E364ATs − , recessiveSide13
    act1-102K359A,E361AWild typeSide16
    act1-103E334A,R335A,K336ALethal, recessiveFront26
    act1-104K315A,E316AWild typeSide18
    act1-105E311A,R312ACs − , Ts − , recessiveFront113
    act1-106R290A,K291A,E292ALethal, recessiveSide41
    act1-107D286A,D288ALethal, partial dominantTop/bottom50
    act1-108R256A,E259ATs − , weakly dominantBack63
    act1-109E253A, R254ALethal, partial dominantFront52
    act1-110E237A,K238ALethal, partial dominantTop/bottom45
    act1-111D222A,E224A,E226ATs − , recessiveSide57
    act1-112K213A,E214A,K215ACs − , Ts − , recessiveFront100
    act1-113R210A,D211AWeak Ts − , recessiveFront21
    act1-115E195A,R196AWild typeTop/bottom11
    act1-116D187A,K191AWild typeBack5
    act1-117R183A,D184AWild typeBack4
    act1-119R116A,E117A,K118ATs − , recessiveBack12
    act1-120E99A,E100ATs − , recessiveSide7
    act1-121E83A,K84ACs − , Ts − , recessiveSide7
    act1-122D80A,D81ACs − , Ts − , recessiveSide9
    act1-123R68A,E72AWild typeBack33
    act1-124D56A,E57ATs − , recessiveFront11
    act1-125K50A,D51ACs − , Ts − , recessiveSide3
    act1-127E270A,D275ALethal, recessiveBack19
    act1-128E241A,D244ALethal, partial dominantTop/bottom40
    act1-129R177A,D179ATs − , recessiveBack10
    act1-131K61A,R62ALethal, partial dominantTop/bottom54
    act1-132R37A,R39ACs − , Ts − , recessiveBack34
    act1-133D24A,D25ACs − , Ts − , recessiveFront34
    act1-135E4AWild typeFront9
    act1-136D2AWild typeND14
    act1-159V159NTs − , recessiveATP cleft17

    CHI, complex haploinsufficiency ND, not determined.

    Most of the act1 alanine scan mutant strains were generated as previously described (Haarer et al. 2007). Strains carrying the act1-107, -108, -127, -128, and -136 alleles were generated by transforming the corresponding heterozygous diploids previously described (Viggiano et al. 2010) with the CEN URA3ACT1 plasmid pKFW29, followed by tetrad dissection to generate the strains used in the complex heterozygosity screens.

    The act1-159 strain was generated by crossing strain DAY245 (MATa leu2his3ura3act1-159:Nat R tub2-201 lyp1can1 used for SGA analysis) to BY4741 an act1-159:Nat R segregant was backcrossed to BY4741 and a haploid act1-159:Nat R segregant from this diploid was transformed with pKFW29 and used in the complex heterozygosity screens. Unlike the other act1 strains, this strain carries the act1-linked tub2-201 mutation.

    Complex heterozygosity screens between act1 alanine scan alleles and the act1∆ CHI gene set

    The complex heterozygosity screens described in this study were performed as previously described (Haarer et al. 2007). To summarize, haploid strains carrying Nat R -marked mutant act1 alleles and also containing ACT1 on a CEN URA3 plasmid (pKFW29) were mated to strains deleted (by the kan R /G418 R marker) for genes previously shown to display complex haploinsufficiency with act1∆ (Haarer et al. 2007) and our unpublished results). Diploids were selected on media containing G418 and nourseothricin, followed by streaking of individual diploid colonies to matched media containing G418 and Nat with or without FOA, which counterselects against cells that carry the URA3 marker of pKFW29. Streaks on +/− FOA media were incubated at 34.5° and 37° for most crosses, or at 25° and 30° for those strains that display temperature sensitivity due to haploinsufficiency of the particular gene deletion being tested. Scoring of the growth defects is as follows: a score of one is lethality, a score of 2 corresponds to severe growth defects as reflected by small colonies as compared to the control heterozygotes, and a score of 3 reflects colony sizes that are perceptibly smaller than the control heterozygotes.

    Interaction degree feature correlation analysis

    The collection of physiological and evolutionary features for the CHI gene set was taken directly from (Costanzo et al. 2010). Pearson correlation was used to measure the correlation between the CHI gene degree and each feature using MATLAB.

    Cluster analysis of the complex heterozygous interactions with actin alleles

    Actin mutant interaction profiles were clustered using Cluster 3.0 (de Hoon et al. 2004). We ran hierarchical clustering for both the alleles and genes sides of the matrix. Similarity was measured using uncentered Pearson correlation with average linkage. The clustering algorithm required the weight of the scores to be reversed: we defined 3 as the strongest score and 1 as the weakest score so that weak scores were closest to non-interactions.

    Molecular modeling

    Molecular models were created using UCSF Chimera (Pettersen et al. 2004) and Adobe Photoshop.

    Fluorescence microscopy

    The Sec4-GFP expressing plasmid pRC556 (gift of Anthony Bretscher) (Schott et al. 2002) was transformed into wild-type haploid strain BY4741, act1-112 haploid strain SVY413, and act1-112/ACT1 wt strain SVY331. Cells were grown to

    2 × 10 7 cells/mL, spotted on cushions of 25% gelatin in synthetic complete medium on glass slides, and static and time-lapse images were captured on a Zeiss Imager.Z1 epifluorescence microscope with a 100X Plan Apochromator objective (oil, numerical aperture of 1.46) using an Orca ER camera (Hamamatsu Photonics). Images were processed with Zeiss AxioVision software and Adobe Photoshop.


    Abstract

    Palivizumab was the first antiviral monoclonal antibody (mAb) approved for therapeutic use in humans, and remains a prophylactic treatment for infants at risk for severe disease because of respiratory syncytial virus (RSV). Palivizumab is an engineered humanized version of a murine mAb targeting antigenic site II of the RSV fusion (F) protein, a key target in vaccine development. There are limited reported naturally occurring human mAbs to site II therefore, the structural basis for human antibody recognition of this major antigenic site is poorly understood. Here, we describe a nonneutralizing class of site II-specific mAbs that competed for binding with palivizumab to postfusion RSV F protein. We also describe two classes of site II-specific neutralizing mAbs, one of which escaped competition with nonneutralizing mAbs. An X-ray crystal structure of the neutralizing mAb 14N4 in complex with F protein showed that the binding angle at which human neutralizing mAbs interact with antigenic site II determines whether or not nonneutralizing antibodies compete with their binding. Fine-mapping studies determined that nonneutralizing mAbs that interfere with binding of neutralizing mAbs recognize site II with a pose that facilitates binding to an epitope containing F surface residues on a neighboring protomer. Neutralizing antibodies, like motavizumab and a new mAb designated 3J20 that escape interference by the inhibiting mAbs, avoid such contact by binding at an angle that is shifted away from the nonneutralizing site. Furthermore, binding to rationally and computationally designed site II helix–loop–helix epitope-scaffold vaccines distinguished neutralizing from nonneutralizing site II antibodies.

    Respiratory syncytial virus (RSV) is a highly contagious human pathogen, infecting the majority of infants before age 2 y, and is the leading cause of viral bronchiolitis and viral pneumonia in infants and children (1, 2). RSV remains a top priority for vaccine development, as thousands of deaths are recorded worldwide each year because of complications from infection (3). To date, there is no licensed RSV vaccine. A major focus of RSV vaccine development has been inclusion of the RSV fusion (F) protein, a class I fusion glycoprotein that is synthesized as a precursor and cleaved into two disulfide-linked fragments upon maturation into a trimer (4). Although the RSV virion contains two additional surface proteins, the highly-glycosylated attachment (G) protein and the small hydrophobic protein, the F protein is highly conserved among strains of RSV strains and is the major target of protective neutralizing antibodies.

    The F protein is known to adopt at least two major conformations: the metastable prefusion conformation and the postfusion conformation. Following attachment of the virion to a cell by the G protein, the F protein undergoes a dramatic structural rearrangement, resulting in fusion of the viral and cell membranes, and in cultured cells causes formation of cell syncytia. Four major neutralizing antigenic regions have been identified to date in the F protein, generally designated antigenic sites I, II, IV, and Ø, with the latter present only in the prefusion conformation. Site II is the target of palivizumab (5), a prophylactic treatment licensed for use in high-risk infants during the RSV season. An RSV F protein subunit vaccine candidate comprising aggregates of the postfusion conformation of RSV F is being tested currently in clinical trials (6), and serum antibody competition with palivizumab has been proposed as a potential serologic correlate of immunity for that vaccine (7, 8). We and others have isolated and studied RSV F-specific mAbs using murine hybridomas (9), sorted macaque B cells (10), and transformed human B cells or human antibody gene phage-display libraries (11, 12). Examples include mAbs 101F (9), D25 (13), and the next-generation site II mAb motavizumab (14). However, there are no reported naturally occurring human mAbs to site II, and palivizumab is an engineered humanized version of the murine mAb 1129 (15). Therefore, the repertoire of human antibodies interacting with site II and the structural basis for their recognition of this major antigenic site is poorly understood.

    To characterize the human immune response to the RSV F protein, we isolated and characterized human mAbs targeting the RSV F protein, and in particular focused discovery efforts on antigenic site II. Defining the structural basis for interaction of site II-specific antibodies revealed new insights into the complexity of this site and diverse modes of recognition that determined whether or not site II competing human antibodies neutralize RSV.


    Summary of Site Directed Mutagenesis

    In brief, point-mutations can be introduced to plasmids using primers (with the desired mutation) in a PCR protocol that amplifies the entire plasmid template. The parent template is removed using a methylation-dependent endonuclease (i.e. DpnI), and bacteria are transformed with the nuclease-resistant nicked plasmid (the PCR product). Plasmids are isolated from the resulting colonies, and screened for the desired modification. Finally, the positive clones are sequenced to confirm the desired modification and the absense of additional modifications.


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    Deliver7-15 days10-15 days15-20days
    Min order24 peptides24 peptides24 peptides
    Peptide modificationsN/C-terminals modification,special amino acids,peptide conjugates, biotin, phosphorylation etc
    Analysis reportCOAHPLC+COA+MSHPLC+COA+MS

    Applications Library typesPurity suggestion
    •Antigen epitope mapping.
    •T-cell epitope identification
    •Protein sequence scanning
    Overlapping LibraryCrude
    •T-cell Immunotherapy
    •Metabolism study of peptide drugs
    Truncation LibraryCrude or ≥70%
    •Antibody epitope mapping
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    Combinatorial Positioning
    Scanning Library
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    Crude or ≥70%
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    Peptide library is a mixture of peptide fragment of certain length, which contains all possible arrangement information of amino acids .There are two kinds of peptide library, one is synthetic peptide library, the other is phage peptide library.The fragment in the peptide library has the property of binding to the corresponding specific protein,A single specific fragment can be selected from the peptide library by using the labeled specific protein,The markers which used for labeling proteins can be enzymes, fluorescein, biotin, isotopes, We call this labeled specific protein as probe.

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    Scrambled Peptide Library

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    T-cell Truncated Library allows high-resolution identification of T cell epitopes across a protein.The peptide was gradually cut from both ends to form peptide library, and then the peptide library was mixed into peptide pool to screen the best epitope of T cell. T cell truncated peptide library provides a powerful guarantee for screening the best epitope of T cell of interest.

    Application area: Test all possible T-cell epitopes in protein

    Combinatorial Positional Scanning Peptide Library

    Combinatorial Positional scanning library is an important tool for optimization of peptide sequences.and is an upgrade to positional scanning.After screening out the important sites affecting activity by alanine library,the modification schemes to improve the activity of peptides can be quickly discovered by varying systematically 2 or more positions at a time(replace each site of the peptide with unnatural/natural amino acids,D-amino acids,Phe/Tyr derivatized amino acids,Phosphorylation/N-Methylation etc)

    It will be 20×20 = 400 peptides to be produced in the process of 2-Positional Scanning with 20 kinds of natural amino acids, 3-Positional Scanning would be 8000 peptides total.Due to the huge work of amino acid substitution, our self developed AuaPepTM Synthesis platform can help you to quickly generate this matrix combination. Please tell us the sequence, the site which to be studied, the purity and quantity, our synthesis platform will automatically help you to generate this matrix.

    Application area: Improvement for peptide biological activity.,Study on the activity of enzyme substrates.

    Omizzur has introduced high-throughput automatic peptide synthesis equipment with independently developed OMIZZUTM synthesis platform to produce mg level and multi sequence peptide products to meet the peptide library demand of clients. Omizzur ’s software will automatically help you build peptide library when you let us the know the sequence, purity and other related requirements you want to study,and the results will be sent back to you by email together with the quotation for your references.

    1.Smith G P. Surface Display and Peptide Libraries. Gene,1993,128.1-2

    2.Devlin JJ,Pangniban L C ,Devlin P E. Random Peptide Libraries:A Sourse of Specific Protein Binding Molecules Science,1990,249:404-406

    3.Scott JK,Smith G P.Searching for Peptide Ligands with an Epitope Library.Science,1990,249:386-390

    4.Houghten RA,Pinilla C,Blondelle SE,et al,Generation and use of synthetic peptide combinatorial libraries for basic research and drug discovery [J].Nature,1991,354:84-86

    5.Ohlmeyer MH,Swanson RN,Dillard LW,et al,Complex synthetic chemical libraries indexed with molecular tags[J],Proc Natl Acad Sci,1993,90:10922-10926


    Conclusions

    ABS-Scan webserver can provide valuable insights on molecular recognition involving protein-ligand interactions. Experimentally determined protein-ligand structures can be studied to understand individual residue contributions towards ligand binding. Modeled complexes can also be submitted to infer the feasibility of the interaction. We believe that ABS-Scan would add one more dimension to the analysis of binding sites in proteins, comparison of various ligand interactions and be of importance to researchers performing ASM studies.


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