Generation of monoclonal antibodies for the assessment of protein purification by recombinant ribosomal coupling

We recently described a conceptually novel method for the purification of recombinant proteins with a propensity to form inclusion bodies in the cytoplasm of Escherichia coli. Recombinant proteins were covalently coupled to the E. coli ribosome by fusing them to ribosomal protein 23 (rpL23) followed by expression in an rpL23 deficient strain of E. coli. This allowed for the isolation of ribsomes with covalently coupled target proteins which could be efficiently purified by centrifugation after in vitro proteolysis at a specific site incorporated between rpL23 and the target protein. rpL23-GFP-His is among the fusion proteins used in our previous study for ribosomal coupling of C-terminally His-tagged green fluorescent protein. To assess the efficiency of separation of target protein from ribosomes, by site-specific proteolysis, we required monoclonal antibodies directed against rpL23 and GFP. We therefore purified rpL23-GFP-His, rpL23-His and GFP from E. coli recombinants using affinity, ion exchange and hydrophobic interaction chromatography. These proteins could be purified with yields of 150, 150 and 1500 microg per gram cellular wet weight, respectively. However, rpL23-GFP-His could only be expressed in a soluble form and subsequently purified, when cells were cultivated at reduced temperatures. The purified rpL23-GFP-His fusion protein was used to immunize balb/c mice and the hybridoma cell lines resulting from in vitro cell fusion were screened by ELISA using rpL23-His and GFP to select for monoclonal antibodies specific for each protein. This resulted in 20 antibodies directed against rpL23 and 3 antibodies directed against GFP. Antibodies were screened for isotypes and their efficiency in western immunoblots. The most efficient antibody against rpL23 and GFP were purified by Protein G Sepharose affinity chromatography. The purified antibodies were used to evaluate the separation of ribosomes from GFP, streptavidin, murine interleukin-6, a phagedisplay antibody and yeast elongation factor 1A by centrifugation, when ribosomes with covalently coupled target protein were cleaved at specific proteolytic cleavage sites. We conclude that the generated antibodies can be used to evaluate ribosomal coupling of recombinant target proteins as well as the efficiency of their separation from the ribosome.     

Recombinant renewable polyclonal antibodies

Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products.     

Recombinant renewable polyclonal antibodies

Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products.     

Genetic immunization for the recovery and purification of recombinant proteins

A system that uses genetic immunization for recombinant protein recovery and purification is described. The genetic sequence encoding a target protein is subcloned into both a eukaryotic and a prokaryotic vector. With the eukaryotic construct, a rabbit is genetically immunized and specific polyclonal antibodies to the encoded protein raised. The prokaryotic construct is used for bacterial transformation and expression of recombinant protein. Recovery and purification of target recombinant protein are obtained by passing the lysate of expressing bacteria through an immunoaffinity column prepared with the polyclonal antibodies raised in the genetically immunized animal. This method allows purification of recombinant protein without fusion tails and can be applied to purify any protein whose encoding genetic sequence is known.     

Determination of complementary antibody pairs using protein A capture with the AlphaScreen assay format

The utility of antibody reagents for the detection of specific cellular targets for both research and diagnostic applications is widespread and continually expanding. Often it is useful to develop specific antibodies as reagent pairs that distinguish different epitopes of the target such that sandwich enzyme-linked immunosorbent assay can be used for selective and specific detection. However, the identification of pairing antibodies is often cumbersome and labor-intensive even with the use of designed peptide-specific epitopes as antigens. We have developed a robust and high-throughput method for identifying pairing complementary antibodies derived either from commercial sources or during a rabbit hybridoma monoclonal screening and selection process using protein A capture with the AlphaScreen bead-based assay format. We demonstrate the value and effectiveness of this assay with three protein targets: Akt2, ATF3, and NAEβ (the β-subunit of the neddylation activation enzyme).     

HCV E2蛋白诱导的体液免疫及CTL应答研究

应用PCR方法扩增出HCVE2基因编码417a．a-750a．a的DNA片段,克隆到原核表达载体pQE30 LacZ启动子下游,转化JM109菌株。在JM109菌株中诱导表达出N端含6个组氨酸的E2融合蛋白,用Ni-NTA-Superflow亲和层析柱纯化作为抗原免疫实验兔和BALB／c鼠。定期取兔血,采用间接ELISA方法检测兔子体内针对E2的抗体水平和维持规律。结果显示,距初次免疫14d兔子体内已有抗体产生,直至免疫第55d抗体水平持续上升,之后抗体水平保持稳定,抗体滴度达到1：3200。六周后,取鼠脾脏制各淋巴细胞,定向刺激扩增后与经过重组真核表达质粒pCE2转染的P815细胞作用,利用LDH释放试验检测作用效果。在E：T＝200：1的情况下,杀伤率超过30％。这些结果表明工程菌株表达的HCVE2蛋白具有良好的免疫原性,可以诱发免疫实验动物机体产生较高滴度的抗体及特异性CTL应答。由此我们认为E2蛋白是发展HCV预防工程蛋白疫苗的合适候选者。     

Detection of TAP-tagged proteins in Western blot, confocal laser scanning microscopy and FACS using the ZZ-domain

Tandem affinity purification of protein complexes has become an important tool in the field of proteomic research. Analysis of the proper intracellular localization of TAP-tagged proteins by immunohistochemistry by specific antibodies is often impossible due to the simultaneous detection of the endogenously synthesized native protein. Here we show that the highly specific interaction of the ZZ-domain of Protein A, which constitutes part of the original TAP-tag, to rabbit IgGs can be used to detect TAP-tagged proteins in fixated cells by Confocal Laser Scanning Microscopy just by the use of labeled secondary antibodies. In addition, such interactions can be exploited for the analysis of transfected cells in FACS and Western blot experiments. Thus, we present valuable tools for the analysis of recombinant proteins on the basis of IgG-ZZ interactions, which can be used even if target specific first antibodies are not available or lack sufficient specificity.     

The remarkable flexibility of the human antibody repertoire; isolation of over one thousand different antibodies to a single protein, BLyS

It is well established that the humoral immune response can generate antibodies to many different antigens. The antibody diversity required to achieve this is believed to be substantial. However, the extent to which the immune repertoire can generate structural diversity against a single target antigen has never been addressed. Here, we have used phage display to demonstrate the extraordinary capacity of the human antibody repertoire. Over 1000 antibodies, all different in amino acid sequence, were generated to a single protein, B-lymphocyte stimulator (BLyS™ protein). This is a highly diverse panel of antibodies as exemplified by the extensive heavy and light chain germline usage: 42/49 functional heavy chain germlines and 19/33 V_λ and 13/35 V_κ light chain germlines were all represented in the panel of antibodies. Moreover, a high level of sequence diversity was observed in the V_H CDR3 domains of these antibodies, with 568 different amino acid sequences identified. Thus we have demonstrated that specific recognition of a single antigen can be achieved from many different VDJ combinations, illustrating the remarkable problem-solving ability of the human immune repertoire. When studied in a biochemical assay, around 500 (40%) of these antibodies inhibited the binding of BLyS to its receptors on B-cell lines. The most potent antibodies inhibited BLyS binding with sub-nanomolar IC₅₀ values and with sub-nanomolar affinities. Such antibodies provide excellent choices as candidates for the treatment of BLyS-associated autoimmune diseases.     

Immunization against proprotein convertase subtilisin-like/kexin type 9 lowers plasma LDL-cholesterol levels in mice

Successful development of drugs against novel targets crucially depends on reliable identification of the activity of the target gene product in vivo and a clear demonstration of its specific functional role for disease development. Here, we describe an immunological knockdown (IKD) method, a novel approach for the in vivo validation and functional study of endogenous gene products. This method relies on the ability to elicit a transient humoral response against the selected endogenous target protein. Anti-target antibodies specifically bind to the target protein and a fraction of them effectively neutralize its activity. We applied the IKD method to the in vivo validation of plasma PCSK9 as a potential target for the treatment of elevated levels of plasma LDL-cholesterol. We show that immunization with human-PCSK9 in mice is able to raise antibodies that cross-react and neutralize circulating mouse-PCSK9 protein thus resulting in increased liver LDL receptor levels and plasma cholesterol uptake. These findings closely resemble those described in PCSK9 knockout mice or in mice treated with antibodies that inhibit PCSK9 by preventing the PCSK9/LDLR interaction. Our data support the IKD approach as an effective method to the rapid validation of new target proteins.     

Specific killing of human melanoma cells with an efficient 10B-compound on monoclonal antibodies

We previously established methods which have enabled us to target a sufficient number of 10B atoms on human melanoma cells to destroy them by thermal neutron irradiation. Monoclonal antibodies were here used as vector of 10B atoms on the target cell. Thermal neutrons require at least 10(9) 10B atoms to destroy the cell. In order to accumulate an adequate number of 10B atoms on target cells, our first approach was to make an effective compound that contains 12 atoms of 10B in a molecule. The second step was to conjugate the compound with an avidin molecule (10B12-avidin). One molecule of the 10B12-avidin carries about 30 atoms of 10B. This 10B12-avidin can be specifically targeted on human melanoma cells by biotinated monoclonal antibodies specific for the cells. Furthermore, the number of 10B atoms on target cells can be augmented by a hapten-antihapten monoclonal antibody system. The cultured human melanoma cells treated with these methods were damaged by thermal neutron irradiation. This is the first study that indicates thermal neutrons do injure target cells boronated by monoclonal antibodies.     

狼疮La蛋白的分子生物学研究进展

狼疮La蛋白,又叫La核糖核蛋白、La自身抗原或干燥综合征B型抗原,是原发性干燥综合征的特异性自身抗原之一。La蛋白拥有多种结构和运输元件,可定位于不同的亚细胞部位与RNA相互作用。通常I丑蛋白主要存在于细胞核内,作为RNA聚合酶Ⅲ的转录因子,与RNA聚合酶Ⅲ转录新生产物结合,调节其转录终止,在转录后发挥分子伴侣作用,稳定RNA前体并促进其正确折叠,帮助其进行加工处理。La蛋白还可以与一类拥有内部核糖体进入位点的细胞内mRNA和病毒RNA结合,调节这类RNA的表达翻译;也可在细胞凋亡时被颗粒酶B酶解,产生特异性酶解片段,诱导特异性抗La自身抗体和干燥综合征的生成。我们就I丑蛋白的分子生物学方面的近期研究进展进行综述。     

Selection of antibody fragments specific for an alpha-helix region of acylphosphatase

The native state of common-type acylphosphatase (AcP) elicits two alpha-helices spanning residues 22-32 and 55-67 in the protein sequence. A peptide corresponding to the second alpha-helix (helix-2) of the protein was used to select phage antibodies consisting of a single chain fragment variable. The selection was performed in the presence of trifluoroethanol, a cosolvent known to induce the formation of helical structure in peptides and proteins. Phage scFv antibodies capable of binding the peptide specifically in a trifluoroethanol-induced alpha-helical conformation were isolated by affinity selection (biopanning). Some of these scFvs were also able to bind the native protein but not the peptide in a non-helical unstructured state. This indicates that the structural determinant recognized by the selected antibodies is the alpha-helical conformation of this specific region, rather than simply its amino acid sequence. This study shows that phage display libraries can be used to raise antibodies one can use as reagents able to target regions of a protein with a specific native-like secondary structure.     

Detection of protein-protein interactions using nonimmune IgG and BirA-mediated biotinylation

Detection of protein-protein interactions in cells is crucial for understanding the biological functions of proteins, including their roles in signal transduction. However, current methods require specific antibodies both for immunoprecipitation and detection, making them expensive and sometimes unreliable. Here we describe protocols for protein-protein interaction assays that use nonimmune IgG-conjugated Sepharose to precipitate the IgG binding domain (ZZ) fused to the bait protein; the interaction partner is fused to Avitag and biotinylated by BirA so that it can be detected by a one-step blot with Dylight 680 streptavidin to detect the Avitag fusion protein. Since this method does not require specific antibodies and is inexpensive, sensitive, and reliable, it should be useful for detecting protein-protein interactions in cells.     

Detection of specific DNA sequences using antibodies recognizing UV-labelled DNA.

This non-isotopic method for detection of nucleic acids is based on the in situ labelling of the nucleic acid by exposure to UV-irradiation. The different UV-induced photoproducts, mainly of the thymidine dimer type, are recognized by purified rabbit antibodies specific to the lesions introduced. The UV-labelled nucleic acids can then be visualized by conventional immunostaining procedures. A major advantage of the technique is the low cost and the ease by which the DNA is specifically labelled. The purified rabbit antibodies were shown to be specific for UV-irradiated DNA, and the method was applied for detection of specific DNA sequences hybridized to homologous target DNA on membrane support. We believe that the sensitivity of the method can be improved, and the significance of using different UV-doses, immunostaining methods and membrane types is discussed.     

Rapid modification of antibodies on the surface of liposomes composed of high-affinity protein A-conjugated phospholipid for selective drug delivery

Antibody-modified liposomes, immuno-liposomes, can selectively deliver encapsulated drug ‘cargos’ to cells via the interaction of cell surface proteins with antibodies. However, chemical modification of both the antibodies and phospholipids is required for the preparation of immuno-liposomes for each target protein using conventional methods, which is time-consuming. In the present study, we demonstrated that high-affinity protein A- (Protein A-R28: PAR28) displaying liposomes prepared by the post-insertion of PAR28-conjugated phospholipid through polyethylene glycol (PEG)-linkers (PAR28-PEG-lipo) can undergo rapid modification of antibodies on their surface, and the liposomes can be delivered to cells based on their modified antibodies. Anti-CD147 and anti-CD31 antibodies could be modified with PAR28-PEG-lipo within 1 h, and each liposome was specifically taken up by CD147- and CD31-positive cells, respectively. The cellular amounts of doxorubicin delivered by anti-CD147 antibody-modified PAR28-PEG-lipo were significantly higher than those of isotype control antibody-modified liposomes. PAR28-PEG-lipo can easily and rapidly undergo modification of various antibodies on their surface, which then makes them capable of selective drug delivery dependent on the antibodies.     

抗iLRP单克隆抗体的制备和鉴定

为了研究抗iLRP (Immature laminin receptor protein)单克隆抗体在免疫治疗中的应用,本研究采用重组iLRP对6~8周龄BALB/c雌性小鼠进行免疫,取免疫后小鼠脾细胞与骨髓瘤细胞系SP2/0进行细胞融合,在HAT (Hypoxanthine aminopterin thymidine)半固体培养基上挑选分离明显的细胞克隆于96孔板培养后,取上清液通过ELISA法进行克隆筛选,初次筛选得到23株阳性克隆,复筛后得到13株稳定分泌抗体的细胞株。抗体类别鉴定显示,其中9株为IgM,3株为Ig G1,1株为IgG2a,轻链类型都为κ型。其中1株IgG1和IgG2a用于制备小鼠腹水,经Protein-G柱纯化,SDS-PAGE检测抗体纯度,Western blotting验证抗体特异性。结果表明,抗体纯度均大于90%,并与i LRP有明显的特异性结合。此结果为后续的深入研究提供了一套完整有效的抗iLRP单克隆抗体生产和鉴定的程序,对以iLRP为治疗靶点的单克隆药物研究以及最近兴起的CAR-T (Chimeric antigen receptor modified T cell)免疫治疗具有积极的意义。     

土拉弗朗西斯菌两种不同长度的外膜蛋白FopA抗原和抗体的制备

目的:根据外膜蛋白 FopA 的序列信息,建立土拉弗朗西斯菌 FopA蛋白全长(FopA-L)和部分(FopA-S)的特异性抗原的 BL21 表达系统,获得高活性的重组 FopA-L、FopA-S蛋白并制备相应的多克隆抗体,为土拉菌的监测、诊断和治疗提供依据。方法:通过 pET100 质粒构建FopA-L及FopA-S的表达载体,转化大肠杆菌BL21细胞并诱导表达FopA-L及FopA-S蛋白,螯合镍离子次氨基三乙酸(Ni-NTA)亲合层析纯化FopA蛋白,用重组蛋白免疫大耳白兔制备多克隆抗体,通过 ELISA、Western 印迹、胶体金免疫层析技术等方法进行检测。结果:构建了FopA-L及FopA-S的表达载体,获得相应的高表达目的蛋白 BL21 细胞株,用表达的蛋白为抗原成功制备了 FopA特异性的抗体,效价皆在1 ∶100000以上且特异性良好。结论:FopA-S与FopA-L两种抗原和相应抗体的制备为建立土拉菌快速检测方法奠定了基础。     

Using Phage and Yeast Display to Select Hundreds of Monoclonal Antibodies: Application to Antigen 85, a Tuberculosis Biomarker

Background

Current diagnostic methods for tuberculosis (TB), a major global health challenge that kills nearly two million people annually, are time-consuming and inadequate. During infection a number of bacterial molecules that play a role in the infective process are released and have been proposed as biomarkers for early TB diagnosis. Antigen 85 (Ag85) is the most abundant secreted TB protein, and a potential target for this diagnostic approach. One of the bottlenecks in the direct detection of such bacterial targets is the availability of robust, sensitive, specific antibodies.

Methods

Using Ag85 as a model, we describe a method to select antibodies against any potential target using a novel combination of phage and yeast display that exploits the advantage of each approach.

Results

The efficiency of this approach was attested to by the 111 specific antibodies identified in initial screens. These were assessed for binding to the different Ag85 subunits, affinity, and activity in sandwich assays.

Conclusions

The novelty of this approach lies in the possibility of screening the entire output of a phage antibody selection in a single experiment by yeast display. This can be considered analogous to carrying out a million ELISAs. The monoclonal antibodies (mAbs) identified in this way show high binding affinity and selectivity for the antigens and offer an advantage over traditional mAbs produced by relatively expensive and time consuming techniques. This approach has wide applicability, and the affinity of selected antibodies can be significantly improved, if required.