Selection Dynamic of Escherichia coli Host in M13 Combinatorial Peptide Phage Display Libraries

Phage display relies on an iterative cycle of selection and amplification of random combinatorial libraries to enrich the initial population of those peptides that satisfy a priori chosen criteria. The effectiveness of any phage display protocol depends directly on library amino acid sequence diversity and the strength of the selection procedure. In this study we monitored the dynamics of the selective pressure exerted by the host organism on a random peptide library in the absence of any additional selection pressure. The results indicate that sequence censorship exerted by Escherichia coli dramatically reduces library diversity and can significantly impair phage display effectiveness.     

噬菌体短肽库的构建及其随机短肽的多样性

噬菌体短肽库是将随机合成的寡核苷酸序列通过与单链噬菌体外壳蛋白基因融合,从而将随机短肽表达于噬菌体的表面。将体外随机化学合成的寡聚核苷酸序列重组到单价噬菌体表达载体,构建了噬菌体短肽库,证明其库容为２×１０￣７集落形成单位（ｃｆｕ）,重组率为９３％。同时将１１个随机克隆进行序列测定,证实其寡聚核苷酸序列和氨基酸的分布几乎是完全随机的,其多样性可以满足特异性短肽筛选的要求。     

Phage display: Concept,innovations, applications and future

Phage display is the technology that allows expression of exogenous (poly)peptides on the surface of phage particles. The concept is simple in principle: a library of phage particles expressing a wide diversity of peptides is used to select those that bind the desired target. The filamentous phage M13 is the most commonly used vector to create random peptide display libraries. Several methods including recombinant techniques have been developed to increase the diversity of the library. On the other extreme, libraries with various biases can be created for specific purposes. For instance, when the sequence of the peptide that binds the target is known, its affinity and selectivity can be increased by screening libraries created with limited mutagenesis of the peptide. Phage libraries are screened for binding to synthetic or native targets. The initial screening of library by basic biopanning has been extended to column chromatography including negative screening and competition between selected phage clones to identify high affinity ligands with greater target specificity. The rapid isolation of specific ligands by phage display is advantageous in many applications including selection of inhibitors for the active and allosteric sites of the enzymes, receptor agonists and antagonists, and G-protein binding modulatory peptides. Phage display has been used in epitope mapping and analysis of protein-protein interactions. The specific ligands isolated from phage libraries can be used in therapeutic target validation, drug design and vaccine development. Phage display can also be used in conjunction with other methods. The past innovations and those to come promise a bright future for this field.     

人源天然ScFv噬菌体抗体库的构建及鉴定

噬菌体抗体库技术是获得治疗性抗体的一条重要途径。以20份健康人外周血为样本,通过提取淋巴细胞、逆转录－PCR（RT PCR）、抗体可变区基因的扩增、重叠PCR获得单链抗体（ScFv）基因,将ScFv克隆入噬粒载体,通过近300次的电转化获得了库容量为1.3×109的全人源天然ScFv噬菌体抗体库。通过随机挑克隆测序和用5种不同抗原筛选对抗体库进行了初步验证。随机测序表明抗体库具有较好的多样性,用5种不同抗原对其进行筛选,均获得了特异性噬菌体抗体的不同富集,表明成功构建了一个多样性良好的人源天然ScFv噬菌体抗体库。     

Trinucleotide cassettes increase diversity of T7 phage-displayed peptide library

Background  

Amino acid sequence diversity is introduced into a phage-displayed peptide library by randomizing library oligonucleotide DNA. We recently evaluated the diversity of peptide libraries displayed on T7 lytic phage and M13 filamentous phage and showed that T7 phage can display a more diverse amino acid sequence repertoire due to differing processes of viral morphogenesis.     

Diversity of the Antibody Response to Tetanus Toxoid: Comparison of Hybridoma Library to Phage Display Library

Monoclonal antibodies are important tools in research and since the 1990s have been an important therapeutic class targeting a wide variety of diseases. Earlier methods of mAb production relied exclusively on the lengthy process of making hybridomas. The advent of phage display technology introduced an alternative approach for mAb production. A potential concern with this approach is its complete dependence on an in vitro selection process, which may result in selection of V_H-V_L pairs normally eliminated during the in vivo selection process. The diversity of V_H-V_L pairs selected from phage display libraries relative to an endogenous response is unknown. To address these questions, we constructed a panel of hybridomas and a phage display library using the spleen of a single tetanus toxoid-immunized mouse and compared the diversity of the immune response generated using each technique. Surprisingly, the tetanus toxoid-specific antibodies produced by the hybridoma library exhibited a higher degree of V_H-V_L genetic diversity than their phage display-derived counterparts. Furthermore, the overlap among the V-genes from each library was very limited. Consistent with the notion that accumulation of many small DNA changes lead to increased antigen specificity and affinity, the phage clones displayed substantial micro-heterogeneity. Contrary to previous reports, we found that antigen specificity against tetanus toxoid is encoded by both Vκ and V_H genes. Finally, the phage-derived tetanus-specific clones had a lower binding affinity than the hybridomas, a phenomenon thought to be the result of random pairing of the V-genes.     

Engineering dihydropteroate synthase (DHPS) for efficient expression on M13 phage

Phage display is a commonly used selection technique in protein engineering, but not all proteins can be expressed on phage. Here, we describe the expression of a cytoplasmic homodimeric enzyme dihydropteroate synthetase (DHPS) on M13 phage, established by protein engineering of DHPS. The strategy included replacement of cysteine residues and screening for periplasmic expression followed by random mutagenesis and phage display selection with a conformation-specific anti-DHPS antibody. Cysteine replacement alone resulted in a 12-fold improvement in phage display of DHPS, but after random mutagenesis and three rounds of phage display selection, phage display efficiency of the library had improved 280-fold. Most of the selected clones had a common Asp96Asn mutation that was largely responsible for the efficient phage display of DHPS. Asp96Asn affected synergistically with the cysteine replacing mutations that were needed to remove the denaturing effect of potential wrong disulfide bridging in phage display. Asp96Asn alone resulted in a 1.8-fold improvement in phage display efficiency, but in combination with the cysteine replacing mutations, a total of 130-fold improvement in phage display efficiency of DHPS was achieved.     

A DNA-binding peptide from a phage display library

A DNA-binding peptide was selected from a random peptide phage display library. For competitive elution using the DNA methyltransferase M.TaqI in the selection step, a biotin-labeled duplex oligodeoxyribonucleotide containing the 5'-TCGA-3' recognition sequence of M.TaqI was employed. Nine of ten phages selected were found to have the same deduced amino acid sequence SVSVGMKPSPRP. The selected phage binds to DNA, as demonstrated in an ELISA.     

Diversity visualization by endonuclease: a rapid assay to monitor diverse nucleotide libraries

Many experiments require a fast and cost-effective method to monitor nucleic acid sequence diversity. Here we describe a method called diversity visualization by endonuclease (DiVE) that allows rapid visualization of sequence diversity of polymerase chain reaction (PCR) products based on DNA hybridization kinetics coupled with the activity of a single-strand specific nuclease. The assay involves only a limited number of steps and can be performed in less than 4 h, including the initial PCR. After PCR, the homoduplex double-stranded DNA (dsDNA) is denatured and reannealed under stringent conditions. During the reannealing process, incubation with S1 nuclease removes single-stranded loops of formed heteroduplexes and the resulting digest is visualized on agarose gel. The sequence diversity is inversely proportional to the band intensities of S1 nuclease surviving dsDNA molecules of expected size. As an example, we employed DiVE to monitor the diversity of panning rounds from a single-framework, semisynthetic single-chain antibody fragment (scFv) phage display library. The results are in good agreement with the observed decrease in diversity in phage display panning rounds toward the selection of monoclonal scFv. We conclude that the DiVE assay allows rapid and cost-effective monitoring of diversities of various nucleotide libraries and proves to be particularly suitable for scaffold-based randomized libraries.     

驼源天然单域重链抗体库的构建与鉴定

从未经主动免疫的健康羊驼(Lama pacos)外周血淋巴细胞中提取总RNA,反转录后作为第一轮PCR的模板。根据重链抗体保守区域设计引物,经巢式PCR法扩增获得了全套重链抗体可变区基因,将其克隆至噬菌粒pHEN1,电转化大肠杆菌TG1得到初级抗体库NAL,含有2×10⁷个独立克隆,菌落PCR和Hinf I酶切分析结果显示,克隆效率大于97%,文库的多样性良好。辅助噬菌体救援后,得到噬菌体展示文库命名为NA-PDL,滴度达10¹³CFU/ml。以真菌毒素人工抗原DON-MBSA为目标抗原,对NA-PDL进行了淘选,第二轮洗脱物中,阳性克隆率达36.4%,提示针对目标抗原的噬菌体颗粒得到了有效富集,文库NA-PDL多样性较好,为后续淘选针对特定抗原的单域重链抗体奠定了基础。     

Efficient selection of DARPins with sub-nanomolar affinities using SRP phage display

There is an ever-increasing demand to select specific, high-affinity binding molecules against targets of biomedical interest. The success of such selections depends strongly on the design and functional diversity of the library of binding molecules employed, and on the performance of the selection strategy. We recently developed SRP phage display that employs the cotranslational signal recognition particle (SRP) pathway for the translocation of proteins to the periplasm. This system allows efficient filamentous phage display of highly stable and fast-folding proteins, such as designed ankyrin repeat proteins (DARPins) that are virtually refractory to conventional phage display employing the post-translational Sec pathway. DARPins comprise a novel class of binding molecules suitable to complement or even replace antibodies in many biotechnological or biomedical applications. So far, all DARPins have been selected by ribosome display. Here, we harnessed SRP phage display to generate a phage DARPin library containing more than 10¹⁰ individual members. We were able to select well behaved and highly specific DARPins against a broad range of target proteins having affinities as low as 100 pM directly from this library, without affinity maturation. We describe efficient selection on the Fc domain of human IgG, TNFα, ErbB1 (EGFR), ErbB2 (HER2) and ErbB4 (HER4) as examples. Thus, SRP phage display makes filamentous phage display accessible for DARPins, allowing, for example, selection under harsh conditions or on whole cells. We envision that the use of SRP phage display will be beneficial for other libraries of stable and fast-folding proteins.     

Peptide epitope identification by affinity selection on bacteriophage MS2 virus-like particles

Filamentous phages are now the most widely used vehicles for phage display and provide efficient means for epitope identification. However, the peptides they display are not very immunogenic because they normally fail to present foreign epitopes at the very high densities required for efficient B-cell activation. Meanwhile, systems based on virus-like particles (VLPs) permit the engineered high-density display of specific epitopes but are incapable of peptide library display and affinity selection. We developed a new peptide display platform based on VLPs of the RNA bacteriophage MS2. It combines the high immunogenicity of MS2 VLPs with the affinity selection capabilities of other phage display systems. Here, we describe plasmid vectors that facilitate the construction of high-complexity random sequence peptide libraries on MS2 VLPs and that allow control of the stringency of affinity selection through the manipulation of display valency. We used the system to identify epitopes for several previously characterized monoclonal antibody targets and showed that the VLPs thus obtained elicit antibodies in mice whose activities mimic those of the selecting antibodies.     

脓毒症单核巨噬细胞特异性结合肽的筛选

利用噬菌体随机肽库展示技术,筛选出与脓毒症单核/巨噬细胞特异性结合的短肽,探索脓毒症治疗的新方法.分别以经过脂多糖(lipopolysaccharide, LPS)处理的人外周血单核细胞株(THP-1)细胞作为筛选的靶细胞,以未经LPS处理的THP-1细胞作为非特异性噬菌体吸附细胞,对噬菌体随机环七肽库进行4轮“差减"筛选,经过细胞ELISA验证阳性噬菌体克隆,对获得的阳性克隆进行DNA测序及生物信息学分析,并进一步利用免疫荧光实验,鉴定噬菌体克隆与LPS处理THP-1细胞的结合特异性.4轮筛选后,随机挑取的噬菌体克隆,测序后得到可与LPS处理的THP-1细胞特异性结合肽.对去冗余后的七肽进行Clustal W多序列比对分析和BlastP蛋白同源相似性分析,细胞免疫荧光检测确定获得的噬菌体展示七肽可与LPS处理的THP-1细胞特异性结合.噬菌体随机肽库技术为脓毒症单核/巨噬细胞表面靶位的筛选提供了高效、快捷的筛选体系,实验获得的多肽基序具有高度保守性和细胞特异性,这些多肽的生物活性将是下一步的研究内容.     

Investigation of de novo totally random biosequences, Part I: A general method for in vitro selection of folded domains from a random polypeptide library displayed on phage

This paper reports the initial phase of a research aimed at investigating the folding frequency within a large library of polypeptides generated with a totally random sequence by phage-display technique. Resistance to proteolytic digestion has been used as a first, rudimentary folding criterion. The present paper describes, in particular, the development of a phage-display vector which has a selectable N-terminal affinity tag so that, after controlled proteolysis, the tag is cleaved from the phage. This enables the positive selection of phages that carry proteolytically resistant proteins. To test this system, avian pancreatic polypeptide (APP), one of the smallest proteins with a known structure, was chosen as a model, and its gene was inserted in a plasmid that was then used for phage display. A sequence of three amino acids, corresponding to a substrate for thrombin, was introduced at different locations within the APP sequence without significantly modifying the tertiary structure, as determined by circular dichroism (CD) analysis. These sequences were then used to show that the target tripeptide sequence was protected against proteolysis by the overall folding of the chain. Thus, these results show that the method permits the discrimination between folded and unfolded protein domains displayed on phage. The application of this protocol to a large library of totally random polypeptide chains is discussed as a preliminary to successive work, dealing with the production of totally random polypeptide sequences.     

A DNA-BINDING PEPTIDE FROM A PHAGE DISPLAY LIBRARY

A DNA-binding peptide was selected from a random peptide phage display library. For competitive elution using the DNA methyltransferase M · TaqI in the selection step, a biotin-labeled duplex oligodeoxyribonucleotide containing the 5′-TCGA-3′ recognition sequence of M · TaqI was employed. Nine of ten phages selected were found to have the same deduced amino acid sequence SVSVGMKPSPRP. The selected phage binds to DNA, as demonstrated in an ELISA.     

抗脐带间充质干细胞表面分子噬菌体ScFv抗体库的构建及初步鉴定

目的：利用噬菌体展示技术构建抗脐带间充质干细胞表面分子噬菌体ScFv抗体库。方法：收集P3代培养的UC-MSCs免疫BALB/c小鼠,提取其脾细胞总RNA,RT-PCR扩增全套VH和VL基因片段,将其先后克隆入噬菌粒pSEX81中,构建成完整的噬菌体ScFv抗体库。结果:构建的噬菌体ScFv抗体库的库容为2×107cfu,ScFv插入重组率为93％,BstN1酶切图谱呈不同多样性。ScFv抗体库经3轮初步筛选后插入重组率达100％,3个克隆出现了相同的酶切图谱,并且随着筛选次数的增加,输出/输入比明显提高,这说明抗体库得到了特异性富集。结论：成功地构建了抗脐带间充质干细胞表面分子噬菌体ScFv抗体库,这为将来筛选特异性抗体和进一步用于间充质干细胞表面特异性分子研究奠定了坚实的基础。     

QSYP peptide sequence is selected from phage display libraries by bovine IgG contaminants in monoclonal antibody preparations

A consensus peptide sequence, QSYP, appears as an artifact during the mapping of monoclonal antibodies (MAbs) using a random peptide phage display library. Phage bearing this QSYP sequence were independently selected by four different laboratories screening separate MAb preparations with the same phage library. In each case, the QSYP sequence was selected in addition to a consensus sequence specific to the MAb. Phage that displayed the QSYP sequence were not bound by the MAb of interest, but rather bound to bovine IgG derived from the FBS present in the hybridoma growth media. The implications of this finding for the interpretation of phage library screening results and possible methods for the removal of bovine IgG from MAb preparations are discussed.     

脑膜炎大肠杆菌IbeA蛋白结合肽序列的亲和筛选

应用噬菌体展示肽库技术,以重组的脑膜炎大肠杆菌致病蛋白IbeA作为靶分子,经过吸附-洗脱-扩增-再吸附的亲和筛选,随机挑选亲和力强的噬菌体克隆,进行ELISA、竞争抑制实验和序列测定。结果显示,经3轮淘选后,间接ELISA鉴定得到高亲和性结合IbeA蛋白的15个阳性克隆。竞争抑制实验结果表明,游离IbeA蛋白能竞争抑制噬菌体结合肽克隆与固相包被的IbeA蛋白的结合,其抑制作用随游离IbeA蛋白浓度的降低而减弱。测序结果得到5种阳性噬菌体克隆展示肽序列。上述结果提示以脑膜炎大肠杆菌IbeA蛋白为靶筛选所获得的噬菌体12肽克隆,具有特异性,其结合肽序列呈现相对保守性。建立的从噬菌体随机肽库筛选IbeA蛋白结合肽的方法具有方便、灵活和高效可行的特点。     

Construction of a large phage display antibody library by in vitro package and in vivo recombination

Capacity and diversity are extremely important to the quality of various phage display libraries. In this work, λ phage-based in vitro package was applied to construct a filamentous phage display antibody library so as to enlarge its capacity and introduce more sequence diversity in the final library. In vivo recombination via Cre recombinase/lox sites was also exploited to create V_H/V_L combination diversity based on multivalent package of λ phage packaging extracts on phagemid DNA concatemers. The library constructed with 10 μg concatenated phagemid DNA and ten vials of λ phage packaging extracts was calculated to contain 1.40×10¹⁰ independent clones. Higher capacity can be easily achieved when more materials are consumed. This strategy is somewhat more efficient than prior methods.     

Random peptide libraries displayed on adeno-associated virus to select for targeted gene therapy vectors

Characterizing the molecular diversity of the cell surface is critical for targeting gene therapy. Cell type-specific binding ligands can be used to target gene therapy vectors. However, targeting systems in which optimum eukaryotic vectors can be selected on the cells of interest are not available. Here, we introduce and validate a random adeno-associated virus (AAV) peptide library in which each virus particle displays a random peptide at the capsid surface. This library was generated in a three-step system that ensures encoding of displayed peptides by the packaged DNA. As proof-of-concept, we screened AAV-libraries on human coronary artery endothelial cells. We observed selection of particular peptide motifs. The selected peptides enhanced transduction in coronary endothelial cells but not in control nonendothelial cells. This vector targeting strategy has advantages over other combinatorial approaches such as phage display because selection occurs within the context of the capsid and may have a broad range of applications in biotechnology and medicine.