File Name: isolating and engineering human antibodies using yeast surface display .zip
This protocol describes the process of isolating and engineering antibodies or proteins for increased affinity and stability using yeast surface display. Single-chain antibody fragments scFvs are first isolated from an existing nonimmune human library displayed on the yeast surface using magnetic-activated cell sorting selection followed by selection using flow cytometry. This enriched population is then mutagenized, and successive rounds of random mutagenesis and flow cytometry selection are done to attain desired scFv properties through directed evolution.
- Isolating and engineering human antibodies using yeast surface display
- Prompting Fab Yeast Surface Display Efficiency by ER Retention and Molecular Chaperon Co-expression
- Yeast Surface Display
Isolating and engineering human antibodies using yeast surface display
Metrics details. The classical yeast display technology relies on tethering an engineered protein to the cell wall by genetic fusion to one subunit of a dimeric yeast-mating agglutination receptor complex. This method enables an efficient genotype—phenotype linkage while exploiting the benefits of a eukaryotic expression machinery.
Over the past two decades, a plethora of protein engineering efforts encompassing conventional antibody Fab and scFv fragments have been reported. In this review, we will focus on the versatility of YSD beyond conventional antibody engineering and, instead, place the focus on alternative scaffold proteins and enzymes which have successfully been tailored for purpose with regard to improving binding, activity or specificity.
Directed evolution is a powerful method that involves 1 the random generation of a broad set of protein variants, 2 their production in an expression host, and 3 the subsequent screening for variants with desired novel functionalities [ 1 , 2 , 3 ]. The method was enabled by the emergence of cell surface display techniques that bring proteins of interest into direct contact with potential interaction partners. Notably, the yeast Saccharomyces cerevisiae proved to be an invaluable tool for the generation of large protein libraries, where each variant is displayed in high copy number on the surface of a single cell, thereby converting gene diversity into cell diversity.
Ultra-high throughput yeast library screening has been extensively used in pharma and biotech industry for the screening of large antibody repertoires aimed at isolating variants with therapeutic relevance. This review focuses on directed evolution of alternative scaffolds and enzymes engineered to improved target binding, specificity or activity using yeast surface display.
The versatility of this screening platform will be emphasized by describing many examples of the engineering of non-antibody molecules as well as functional screening strategies for the modification of enzymes. The expression and display of proteins on the surface of bacterial and eukaryotic host cells has become increasingly attractive, as demonstrated by the numerous platform technologies that have been developed [ 4 , 5 , 6 , 7 , 8 ]. In contrast to bacteria, for which potent display methods have been established over the years [ 9 ], eukaryotes offer the additional advantage of an efficient posttranslational modification machinery as well as a quality control mechanism for protein folding that encompasses chaperones and foldases [ 10 ].
In particular, surface display on S. Since strategies for the generation and high-throughput screening of large combinatorial libraries of human antibodies using yeast surface display have been extensively reviewed elsewhere [ 12 ], the focus of this review is placed on the isolation of tailor-made binding proteins as well as enzymes with improved functional characteristics.
This review highlights the versatility of the yeast surface display platform beyond classical antibody engineering and provides an overview of the many engineering approaches that have successfully been conducted with regard to improving not only protein binding but also enzyme activity and specificity.
In general, the principle of microbial cell surface display relies on the establishment of a genotype—phenotype linkage which converts gene diversity into protein diversity. This link is an essential pre-requisite for the success of any surface display screening platform and it is usually realized upon fusing the protein of interest to a microbial cell surface protein. In the case of yeast surface display, a variety of different anchor proteins has been evaluated for efficiently tethering the protein of interest to the cell wall [ 10 ].
The most commonly used anchor is the S. The classical yeast surface display method as pioneered by Boder and Wittrup in [ 4 ] relies on the N -terminal fusion of a protein of interest to Aga2p Fig. Nevertheless, the orientation can be altered upon employing C -terminal fusions, depending on the protein to be displayed, as for some proteins a free N -terminus can be crucial for efficient functionality [ 13 ].
Depending on the cell wall protein that is utilized for immobilization, the number of copies of the protein of interest that are displayed can vary [ 14 ]. When using the Aga2p system, however, it has been demonstrated that up to 10 5 copies of the fusion protein can be displayed on a single cell [ 4 ]. For some proteins, surface display efficiency correlated with protein secretion levels, i. For instance, Kieke and coworkers achieved good surface display levels for a previously display-incompetent single-chain T cell receptor.
Upon combining several stability-enhancing mutations, they improved the display levels from 10, up to 50, copies per yeast cell [ 15 ].
Yeast surface display setup as pioneered by Boder and Wittrup in [ 1 ]. The protein of interest a vNAR domain in this particular depiction is fused to the C -terminus of the Aga2p protein. Aga2p is covalently linked to Aga1p via two disulfide bonds.
Aga1p anchors the fusion protein to the cell wall, ensuring a genotype—phenotype coupling of individual yeast cells. Genetically, the Aga2p fusion protein is encoded on a plasmid and its expression is under the control of a galactose-inducible promotor GAL1. The Aga1p protein on the other hand is encoded in the yeast genome and also controlled by a GAL1 promotor sequence. The assembly of Aga1p and Aga2p is ensured by the formation of two disulfide bonds. For subsequent functional screenings, Boder and Wittrup included epitope tags which were fused to the C -terminus of the protein of interest or inserted between the Aga2p and the protein of interest.
Upon immunofluorescence staining of these tags, full-length protein expression can be verified using a flow cytometer. This offers an additional quality control check during the isolation of variants with desired functionalities and represents a distinct advantage over phage display [ 19 , 20 ]. However, detection of a C -terminal tag does not give information on the structural integrity of the displayed protein. This obstacle can be circumvented upon using a conformation-specific detection antibody for the protein of interest [ 21 ].
After the incubation of yeast cells with the respective target protein, the interaction can be analyzed using fluorescently-labeled detection reagents specifically addressing the target. The generation of yeast libraries for the purpose of identifying a protein variant with superior abilities such as improved stability, affinity or, in case of enzymes, higher catalytic activity usually relies on the mutagenesis of a precursor protein.
Mutations can be introduced through error-prone PCR [ 22 , 23 ], DNA-shuffling [ 24 , 25 ], codon-based randomization [ 26 , 27 ] or structure-guided design [ 28 ].
Subsequently, yeast cells are transformed with the genetic library resulting in genotype—phenotype linked yeast libraries with sizes up to 10 9 transformants. Although several orders of magnitude smaller than libraries generated using phage, ribosomal or mRNA display, the utilization of yeast display offers the inherent advantage of simultaneously analyzing the library content in terms of surface display via the detection of epitope tags and target binding, thereby enabling a functional read-out.
Yeast surface display has emerged as a straightforward strategy for human antibody engineering. This topic has been extensively reviewed and, therefore, will not be highlighted herein [ 10 , 12 , 29 , 30 ]. Beyond antibodies, alternative scaffold-based affinity reagents have emerged as a promising class of biomolecules with therapeutic potential [ 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 ]. These proteins exhibit advantageous properties in comparison to full-length monoclonal antibodies, such as improved tissue penetration, superior stability and cost-efficient production [ 32 , 39 ].
In general, an alternative scaffold protein is capable of exhibiting a variety of amino acid sequences on a constant backbone region [ 40 ]. A pre-requisite that renders a protein an ideal alternative binding scaffold is a certain tolerance towards structural alterations which are necessary in order to tailor the protein for purpose and enable molecular recognition [ 41 , 42 ].
In contrast to conventional antibodies, they are often able to interact with cryptic or hidden epitopes that are difficult to address. As an example, shark-derived vNAR domains as well as camelid-derived VHH domains have been reported to specifically engage the cleft-like catalytic site of enzymes [ 43 , 44 , 45 ].
In addition, recombinant production of these scaffolds is often cheaper compared to the costs of producing monoclonal antibodies, as no posttranslational modifications are required and recombinant expression in Escherichia coli rather than in mammalian cells can be performed. Some alternative binding proteins, such as miniproteins and DARPins, exhibit a resistance towards chemical denaturing or degradation by proteases.
This renders them especially interesting for oral applications, as antibodies and antibody-derived fragments are degraded in the acidic gastrointestinal environment [ 36 ].
However, their efficient passage across epithelial barriers represents an unsolved problem. Alternative binding proteins have been developed for various applications including therapy, diagnostics and imaging. Many of these scaffolds already reached late-stage clinical trials or have been FDA-approved, such as the miniprotein Ziconotide, once again demonstrating their immense potential [ 46 ].
One key aspect that must be considered with regard to therapeutic applications of these scaffolds is their immunogenic potential. However, previous studies have shown that even fully human antibodies can be immunogenic in humans [ 47 ], so a detailed evaluation of the immunogenicity of alternative scaffold proteins needs to be carried out independently [ 36 ].
Most scaffold proteins currently in clinical trials, however, are either derived from human proteins or comprise a low immunogenic profile [ 36 ]. Other scaffolds, such as affibodies, are mostly evaluated for short-lived applications, i. Alternative scaffold proteins have been obtained and engineered using various display techniques and strategies for isolating variants with tailor-made properties.
Specific examples that employed yeast display as high throughput platform are detailed below Fig. For some scaffolds, the eukaryotic expression machinery of yeast cells can be especially advantageous due to the presence of a high number of disulfide bonds as it is the case for miniproteins or Ig-derived scaffolds. Structural depictions of the alternative scaffold proteins discussed in the scope of this review.
The proportions of the depicted scaffold proteins are relative and do not reflect the actual differences in size. Engineering of the 10th type III domain of fibronectin termed Fn3 hereafter in terms of its use as a novel scaffold protein was first described by Koide and coworkers in [ 48 ]. In contrast to other proteins belonging to the IgSF, the Fn3 domain does not comprise any disulfide bonds. Moreover, this monomeric architecture as well as the absence of disulfide bonds allows for a facile expression of Fn3 domains in E.
Koide and coworkers were the first to describe the engineering of Fn3 domains for the purpose of molecular recognition. They elegantly demonstrated that highly specific Fn3 binders targeting ubiquitin could be isolated from a phage-displayed library that was comprised of Fn3 domains with randomized amino acids in two surface-exposed loops. They further characterized the structural integrity of a dominant Fn3 single clone, showing that this variant tolerated 12 mutations out of 94 residues and emphasizing the potential of Fn3 as alternative binding scaffold [ 48 ].
Although their approach involved phage display as the platform technology, it was later shown by Lipovsek and coworkers that Fn3 domains are also compatible with yeast display [ 24 ]. Their engineering approach focused on the generation of several Fn3 yeast libraries with mutations in either one or two loops of the protein scaffold. Both libraries were sampled towards hen egg lysozyme and subsequent affinity maturation of initial binders upon loop shuffling and recursive mutagenesis yielded variants with picomolar affinities.
In a follow-up investigation, Hackel and colleagues further improved the affinity maturation process of yeast-displayed Fn3 domains yielding binders against lysozyme with single-digit picomolar affinities [ 51 ]. Using yeast surface display, Koide and coworkers demonstrated the feasibility of a binary code interface comprising serine and tyrosine residues for the diversification of Fn3 domains [ 52 ].
Their work illustrates that this minimal amino acid diversification approach is a valid strategy not only for obtaining high-affinity Fab fragments as demonstrated previously [ 53 ] but also for acquiring smaller alternative scaffold proteins. The success of this approach seems to stem from the ability of tyrosine residues to form a plethora of different nonbonded interactions as well as from the remarkable conformational diversity of the Fn3 loops that extent the rather limited chemical diversity.
The generation of mutant Fn3 libraries in the yeast as well as phage display format has been achieved not only by randomizing loop residues, but also upon the diversification of amino acid residues present in the protein backbone. In , Hackel and coworkers isolated high-affinity fibronectin domains targeting various epitopes of the epidermal growth factor receptor EGFR using yeast surface display [ 55 ].
Chen and coworkers developed an extensive protocol for the isolation of Fn3 domains from yeast-displayed libraries [ 56 ]. Mann and colleagues used Fn3 domains in combination with yeast surface display for the identification of binders specifically targeting a distinct surface patch of the mitogen activated protein kinase MAPK Erk-2 [ 57 ].
They applied screening procedures including positive and negative selection steps. Positive selection steps relied on wildtype Erk-2 while negative selections encompassed a mutant version of Erk-2, leading to the enrichment of Fn3 domains specifically targeting the desired patch on the kinase surface. In another investigation, Sha and coworkers utilized yeast display screenings for the isolation of Fn3 domains towards the N - and C -terminal SH2 domains of the Src-homology 2 domain-containing phosophatase 2 SHP2 , a subunit of the multiprotein complex of the therapeutically relevant tyrosine kinase BCR-ABL [ 58 ].
Initial libraries were screened using phage display while additional mutagenesis and subsequent translation into the yeast display format yielded the final candidates. To this end, Heinzelman and colleagues aimed for a significant decrease in antigen affinity at an endosomal pH of 5. Such pH-sensitive Fn3 domains might be useful for continuous receptor down-regulation in a therapeutic manner, allowing the release of the fibronectin from its receptor target in the acidic endosome [ 60 ].
Heinzelman and coworkers chose a site-directed mutagenesis approach that focused on the mutation of distinct framework rather than loop residues to histidine. The mutated positions were determined using a structure-guided algorithm.
The resulting variants were analyzed on the yeast surface in terms of pH-sensitive binding to their antigen EGFR and yielded several Fn3 domains with the desired characteristics.
Recently, Park et al. Selected single clones bound human EphA2 with single-digit nanomolar affinities and one candidate was shown to function as an in vivo imaging probe in mouse xenograft models. Conclusively, yeast surface display of fibronectin scaffolds has yielded an array of binders towards various therapeutically relevant targets, often with impressive affinities for their respective antigens.
The approaches described in this section further underline the versatility of this scaffold as well as its therapeutic relevance [ 37 , 50 ]. However, it should be noted that an impressing variety of high-affinity Fn3 domains has also been generated upon employing phage [ 48 , 61 ] and mRNA display [ 62 ]. As Fn3 domains are devoid of any disulfide bonds or glycosylation sites, they are compatible with bacterial surface display formats.
Prompting Fab Yeast Surface Display Efficiency by ER Retention and Molecular Chaperon Co-expression
Protocol DOI: This protocol describes the process of isolating and engineering antibodies or proteins for increased affinity and stability using yeast surface display. Single-chain antibody fragments scFvs are first isolated from an existing nonimmune human. Single-chain antibody fragments scFvs are first isolated from an existing nonimmune human library displayed on the yeast surface using magnetic-activated cell sorting selection followed by selection using flow cytometry. This enriched population is then mutagenized, and successive rounds of random mutagenesis and flow cytometry selection are done to attain desired scFv properties through directed evolution. Labeling strategies for weakly binding scFvs are also described, as well as procedures for characterizing and 'titrating' scFv clones displayed on yeast. The ultimate result of following this protocol is a panel of scFvs with increased stability and affinity for an antigen of interest.
Metrics details. The classical yeast display technology relies on tethering an engineered protein to the cell wall by genetic fusion to one subunit of a dimeric yeast-mating agglutination receptor complex. This method enables an efficient genotype—phenotype linkage while exploiting the benefits of a eukaryotic expression machinery. Over the past two decades, a plethora of protein engineering efforts encompassing conventional antibody Fab and scFv fragments have been reported. In this review, we will focus on the versatility of YSD beyond conventional antibody engineering and, instead, place the focus on alternative scaffold proteins and enzymes which have successfully been tailored for purpose with regard to improving binding, activity or specificity. Directed evolution is a powerful method that involves 1 the random generation of a broad set of protein variants, 2 their production in an expression host, and 3 the subsequent screening for variants with desired novel functionalities [ 1 , 2 , 3 ].
Isolating and engineering human antibodies using yeast surface display (vol 1, pg , ). July ; Nature Protocols 1(2)
Yeast Surface Display
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For antibody discovery and engineering, yeast surface display YSD of antigen-binding fragments Fabs and coupled fluorescence activated cell sorting FACS provide intact paratopic conformations and quantitative analysis at the monoclonal level, and thus holding great promises for numerous applications. Moreover, fusing ER retention sequences ERSs with light chain also enhanced Fab display quality at the expense of display quantity, and the degree of improvements was correlated with the strength of ERSs and was more significant for Infliximab than Adalimumab. The feasibility of affinity maturation was further demonstrated by isolating a high affinity Fab clone from 3 or 5 spiked libraries. Monoclonal antibodies mAbs represent the fastest growing class of therapeutics in the last decades.
Затем, с еще мокрыми волосами, угощал поверженного соперника орешками и соком. Как у всех молодых профессоров, университетское жалованье Дэвида было довольно скромным. Время от времени, когда надо было продлить членство в теннисном клубе или перетянуть старую фирменную ракетку, он подрабатывал переводами для правительственных учреждений в Вашингтоне и его окрестностях. В связи с одной из таких работ он и познакомился со Сьюзан. В то прохладное осеннее утро у него был перерыв в занятиях, и после ежедневной утренней пробежки он вернулся в свою трехкомнатную университетскую квартиру. Войдя, Дэвид увидел мигающую лампочку автоответчика. Слушая сообщение, он выпил почти целый пакет апельсинового сока.
Вот именно! - крикнул Джабба. - Посмотрите уран. Его сверхкритическую массу. - М-м… сто десять фунтов, - сказала Соши. - Сто десять? - оживился Джабба.
Не бывает такой диагностики, которая длилась бы восемнадцать часов. Все это вранье, и ты это отлично знаешь. Скажи мне, что происходит. Сьюзан прищурилась. Ты сам отлично знаешь, что происходит.
В любой другой реальности было бы куда больше здравого смысла. Я, университетский профессор, - подумал он, - выполняю секретную миссию. Бармен с любезной улыбкой протянул Беккеру стакан: - A su gusto, senor. Клюквенный сок и капелька водки.
Если до этого Хейл не знал, что они идут, то теперь отлично это понял.
Пожилой человек вдруг поднялся и куда-то побежал, видимо, вызвать скорую. Танкадо явно терял последние силы, но по-прежнему совал кольцо прямо в лицо тучному господину. Тот протянул руку, взял Танкадо за запястье, поддерживая остававшуюся на весу руку умирающего. Танкадо посмотрел вверх, на свои пальцы, на кольцо, а затем, умоляюще, - на тучного господина. Это была предсмертная мольба.
- Стратмор приподнял брови, точно ждал объяснений.
NSA. GOV Гнев захлестнул ее, но она сдержалась и спокойно стерла сообщение. - Очень умно, Грег. - Там подают отличный карпаччо. - Хейл улыбнулся.
У него не было сомнений относительно того, что произошло: Стратмор совершил ошибку, обойдя фильтры, и теперь пытался скрыть этот факт глупой версией о диагностике. Чатрукьян не был бы так раздражен, если бы ТРАНСТЕКСТ был его единственной заботой. Однако это было не. Несмотря на свой внушительный вид, дешифровальное чудовище отнюдь не было островом в океане. Хотя криптографы были убеждены, что система фильтров Сквозь строй предназначалась исключительно для защиты этого криптографического декодирующего шедевра, сотрудники лаборатории систем безопасности знали правду.
Чатрукьян растерялся. - Так вы обратили внимание. - Конечно.