Filamentous bacteriophage display of a bifunctional protein A::scFv fusion

Filamentous bacteriophage display is a powerful and widely used technology for the selection of affinity ligands. However, the commonly used phagemid systems result in the production of a population of phage of which those displaying the ligand of interest represent only a small proportion. Through simple dilution and nonspecific binding effects, the presence of large numbers of ligand-free phage reduces the likelihood that weak binders will be successfully selected from a ligand library. To provide a means of avoiding such problems, we have introduced an affinity handle into the phage that permits the purification of ligand-displaying phage. The IgG binding domains ofStaphylococcus ciureus protein A (SpA) were fused to a ligand (single chain Fv[scFv]) which is displayed as a fusion with the phage surface protein ApIII. Phage-displaying SpA were separated by affinity chromatography using immobilized human IgG from non-displaying phage and the purified phage were shown to possess functional scFv. Comparisons of fusion proteins in which either the scFv or the affinity handle occupied the amino terminus of the fusion protein showed that, whereas SpA function was unaffected by position, scFv function was compromised when the scFv did not occupy the amino terminus.     

High‐efficiency affinity precipitation of multiple industrial mAbs and Fc‐fusion proteins from cell culture harvests using Z‐ELP‐E2 nanocages

Affinity precipitation using Z‐elastin‐like polypeptide‐functionalized E2 protein nanocages has been shown to be a promising alternative to Protein A chromatography for monoclonal antibody (mAb) purification. We have previously described a high‐yielding, affinity precipitation process capable of rapidly capturing mAbs from cell culture through spontaneous, multivalent crosslinking into large aggregates. To challenge the capabilities of this technology, nanocage affinity precipitation was investigated using four industrial mAbs (mAbs A–D) and one Fc fusion protein (Fc A) with diverse molecular properties. A molar binding ratio of 3:1 Z:mAb was sufficient to precipitate >95% mAb in solution for all molecules evaluated at ambient temperature without added salt. The effect of solution pH on aggregation kinetics was studied using a simplified two‐step model to investigate the protein interactions that occur during mAb–nanocage crosslinking and to determine the optimal solution pH for precipitation. After centrifugation, the pelleted mAb–nanocage complex remained insoluble and was capable of being washed at pH ≥ 5 and eluted with at pH < 4 with >90% mAb recovery for all molecules. The four mAbs and one Fc fusion were purified from cell culture using optimal process conditions, and >94% yield and >97% monomer content were obtained. mAb A–D purification resulted in a 99.9% reduction in host cell protein and >99.99% reduction in DNA from the cell culture fluids. Nanocage affinity precipitation was equivalent to or exceeded expected Protein A chromatography performance. This study highlights the benefits of nanoparticle crosslinking for enhanced affinity capture and presents a robust platform that can be applied to any target mAb or Fc‐containing proteins with minimal optimization of process parameters.     

Expression, purification, and characterisation of nesiritide using an E. coli expression system

Nesiritide, the recombinant human brain natriuretic peptide, is involved in the regulation of cardiovascular homeostasis and has been approved for treatment of patients with congestive heart failure. We prepared a synthetic cDNA construct of Nesiritide to generate a fusion protein with an affinity handle and 41 amino acid peptide of β-galactosidase. The fusion protein was expressed mainly in the inclusion bodies and accounted for approximately 20% of total cellular protein. After purification by Ni-IDA affinity chromatography and renaturation, the fusion protein was cleaved with purified recombinant enterokinase. Nesiritide was purified by pH precipitation/ion exchange chromatography followed by source phenyl chromatography to obtain protein with > 99% purity (determined by RPHPLC) and a mass of 3,464 Daltons. The potency (ED₅₀) of the purified protein was equivalent to that of Natrecor (Innovator formulation). Analytical methods were developed to identify oxidised, reduced and other related impurities. The expression strategy described in this work allows the convenient generation of high yield Nesiritide and enabled ease of purification.     

Antibody-selectable filamentous fd phage vectors: affinity purification of target genes

Foreign DNA fragments can be inserted into a minor coat protein gene of filamentous phage, creating a fusion protein that is incorporated into the virion; we call these particles "fusion phage". The foreign amino acids are displayed on the surface, allowing fusion phage bearing antigenic determinants from a target gene to be purified in infectious form by affinity to antibody directed against the gene product. Here we introduce fusion-phage vectors that accept foreign DNA inserts with little effect on phage function; and describe affinity purification of virions bearing a target determinant from a 10(8)-fold excess of phage not bearing the determinant, using minute amounts of antibody. These "antibody-selectable" vectors are a promising alternative to conventional expression systems for using antibodies to clone genes, though the ability to isolate rare clones from actual libraries remains to be demonstrated.     

Affinity purification of fibrinogen using a ligand from a peptide library.

An affinity resin containing the peptide ligand Phe-Leu-Leu-Val-Pro-Leu (FLLVPL) has been developed for the purification of fibrinogen. The ligand was identified by screening a solid-phase combinatorial peptide library using an immunostaining technique. The specific binding of fibrinogen to the ligand has been characterized by isothermal calorimetry and adsorption isotherms and is dominated by both hydrophobic interactions and ionic interactions with the N-terminal free amino group. The effective association constant of fibrinogen was substantially higher when the peptide was immobilized on the resin than in solution; moreover, it increased with increasing peptide density, suggesting a cooperative binding effect. A low ionic strength buffer at pH 4 was used successfully to elute adsorbed fibrinogen from the column with high purity, retention of factor XIII crosslinking activity, and minimal, if any, loss of biological function. This general approach to ligand selection and characterization can be used to develop peptide ligands for the affinity purification of diverse proteins on a large scale.     

Utilization of Arg-elution method for FLAG-tag based chromatography

FLAG-tag is one of the commonly used purification technologies for recombinant proteins. An antibody, M2, specifically binds to the FLAG-tag whether it is attached to N- or C-terminus of proteins to be purified. The bound proteins are generally eluted by competition with a large excess of free FLAG peptide. This requires synthetic FLAG peptide and also removal of bound FLAG peptide for M2 column regeneration. We have shown before that arginine at mild pH can effectively dissociate protein–protein or protein–ligand interactions, e.g. in Protein-A, antigen and dye-affinity chromatography. We have tested here elution of FLAG-fused proteins by arginine for columns of M2-immobilized resin using several proteins in comparison with competitive elution by FLAG peptide or low pH glycine buffer. Active and folded proteins were successfully and effectively eluted using 0.5–1 M arginine at pH 3.5–4.4, as reported in this paper.     

Rational design of peptide affinity ligands for the purification of therapeutic enzymes

Non‐mAb biologics represent a growing class of therapeutics under clinical development. Although affinity chromatography is a potentially attractive approach for purification, the development of platform technologies, such as Protein A for mAbs, has been challenging due to the inherent chemical and structural diversity of these molecules. Here, we present our studies on the rapid development of peptide affinity ligands for the purification of biologics using a prototypical enzyme therapeutic in clinical use. Employing a suite of de novo rational and combinatorial design strategies we designed and screened a library of peptides on microarray platforms for their ability to bind to the target with high affinity and selectivity in cell culture fluid. Lead peptides were evaluated on resin in batch conditions and compared with a commercially available resin to evaluate their efficacy. Two lead candidates identified from microarray studies provided high binding capacity to the target while demonstrating high selectivity against culture contaminants and product variants compared to a commercial resin system. These findings provide a proof‐of‐concept for developing affinity peptide‐based bioseparations processes for a target biologic. Peptide affinity ligand design and screening approaches presented in this work can also be easily translated to other biologics of interest. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:987–998, 2018     

Intein-mediated protein purification of fusion proteins expressed under high-cell density conditions in E. coli

The intein-mediated purification system has the potential to significantly reduce the recovery costs of industrial recombinant proteins. The ability of inteins to catalyze a controllable peptide bond cleavage reaction can be used to separate a recombinant protein from its affinity tag during affinity purification. Inteins have been combined with a chitin-binding domain to serve as a self-cleaving affinity tag, facilitating highly selective capture of the fusion protein on an inexpensive substrate--chitin (IMPACT) system, New England Biolabs, Beverly, MA). This purification system has been used successfully at a lab scale in low cell density cultures, but has not been examined comprehensively under high-cell density conditions in defined medium. In this study, the intein-mediated purification of three commercially relevant proteins expressed under high-cell density conditions in E. coli was studied. Additionally, losses during the purification process were quantified. The data indicate that the intein fusion proteins expressed under high cell density fermentations were stable in vivo after induction for a significant duration, and the intein fusion proteins could undergo thiol or pH and temperature initiated cleavage reaction in vitro. Thus, the intein-mediated protein purification system potentially could be employed for the production of recombinant proteins at the industrial-scale.     

Development and validation of an affinity chromatography step using a peptide ligand for cGMP production of factor VIII

An affinity chromatography step was developed for purification of recombinant B-Domain Deleted Factor VIII (BDDrFVIII) using a peptide ligand selected from a phage display library. The peptide library had variegated residues, contained both within a disulfide bond-constrained ring and flanking the ring. The peptide ligand binds to BDDrFVIII with a dissociation constant of approximately 1 microM both in free solution and when immobilized on a chromatographic resin. The peptide is chemically synthesized and the affinity resin is produced by coupling the peptide to an agarose matrix preactivated with N-hydroxysuccinimide. Coupling conditions were optimized to give consistent and complete ligand incorporation and validated with a robustness study that tested various combinations of processing limits. The peptide affinity chromatographic operation employs conditions very similar to an immunoaffinity chromatography step currently in use for BDDrFVIII manufacture. The process step provides excellent recovery of BDDrFVIII from a complex feed stream and reduces host cell protein and DNA by 3-4 logs. Process validation studies established resin reuse over 26 cycles without changes in product recovery or purity. A robustness study using a factorial design was performed and showed that the step was insensitive to small changes in process conditions that represent normal variation in commercial manufacturing. A scaled-down model of the process step was qualified and used for virus removal studies. A validation package addressing the safety of the leached peptide included leaching rate measurements under process conditions, testing of peptide levels in product pools, demonstration of robust removal downstream by spiking studies, end product testing, and toxicological profiling of the ligand. The peptide ligand affinity step was scaled up for cGMP production of BDDrFVIII for clinical trials.     

Development of phage biopanning strategies to identify affinity peptide ligands for kappa light chain Fab fragments

In this work, two phage biopanning strategies were developed to identify affinity peptides for a single Fab and multiple kappa Fabs. For the biopanning rounds, protein L beads were employed to bind Fab targets in a fixed orientation, and NHS functionalized magnetic beads were used to facilitate evaluation of low pH elution conditions. The resulting peptide sequences were synthesized and the binding to different Fabs was evaluated using fluorescence polarization. The first biopanning approach yielded a peptide with similar affinities for two forms of the Fab (recombinantly expressed and post papain-digestion) as well as the intact antibody. While moderate affinity was observed toward a murine variant of the Fab with the same complementarity determining regions (CDR) region but different framework, minimal binding occurred to a Fab with high sequence homology but containing different CDR loops. The second biopanning strategy yielded a peptide with affinity for all three kappa Fabs indicating that it may be a good lead for the development of more general affinity reagents for recombinant kappa Fabs. Finally, an affinity peptide column was developed, and its efficacy was demonstrated for Fab purification from a complex cell culture fluid mixture. The results presented in this article demonstrate that different peptide-based phage biopanning strategies can be effectively employed to identify affinity peptide leads for specific Fab and more general kappa Fab purifications.     

Preparation and characterization of scFv for affinity purification of reteplase

The work was to explore the feasibility of protein affinity purification using ligand isolated from phage library. Reteplase was used as the model protein and a humanized semi-synthetic single chain fragment variable phage library as the source of ligand. After four rounds of biopanning, reteplase-specific phage clones were greatly enriched. The scFv gene from the best phage clone was inserted to pET-29a and expressed in E. coli Rosseta. After purification by nickel-affinity and refolding, this scFv protein was proven to recognize reteplase specifically and sensitively in ELISA and dot-blotting. Its binding constant to reteplase was 1.84x10(-8) M, measured by surface plasmon resonance. After immobilized on Sepharose 4B, the scFv was used for the affinity purification of reteplase from milk. It was found that reteplase was highly purified from the starting material. In conclusion, it has been demonstrated that humanized scFv prepared with this approach could be used as a practical affinity ligand for efficient and cost-effective purification of reteplase, as well as other therapeutic proteins.     

Characterization of an Anti-Thioredoxin Monoclonal Antibody

An anti-E. coli thioredoxin monoclonal antibody, IMM-3C6, which showed high specificity to thioredoxin as assessed by indirect ELISA, was generated using hybridoma technology. The affinity constant of IMM-3C6 to thioredoxin was 0.40×10⁹ m⁻¹ and its sensitivity to thioredoxin fusion protein in dot blotting was 50 ng. In sandwich ELISA, it detected thioredoxin fusion protein between 16 and 150 ng/ml. By using IMM-3C6 as the ligand, thioredoxin fusion protein was successfully purified by affinity chromatography. IMM-3C6 was confirmed to be a useful tool for immunoassay and purification of thioredoxin fusion proteins. These authors contributed equally to the work. Received 21 September 2005; Revisions requested 7 October 2005; Revisions received 10 November 2005; Accepted 11 November 2005     

Recovery of a monoclonal antibody from hybridoma culture supernatant by affinity precipitation with Eudragit S-100

An IgG₁ monoclonal antibody (MAB) was isolated from hybridoma culture supernatant by affinity precipitation with an Eudragit S-100-based heterobifunctional ligand. Affinity binding was performed in a homogeneous aqueous phase at pH 7.5 followed by precipitation of the bound affinity complex by lowering the pH to 4.8. After two washing steps, elution of specifically bound MAB was achieved by incubating the precipitate with 0.1 M glycine.HCl pH 2.5. The influence of elution volume and time on the recovery of active MAB and the overall purification factor were studied. The best conditions enabled the recovery of 50.2% of active MAB with a purification factor of 6.2. A further dialysis against 50 mM Tris.HCl pH 8.0 increased the activity yield and the purification factor to 68.4% and 8.3, respectively. This result showed that part of the antibody activity loss during affinity precipitation was due to a reversible inactivation process, being easily recovered after a refining dialysis step.     

Design, construction and function of a multicopy display vector using fusions to the major coat protein of bacteriophage M13.

Incorporation of numerous copies of a heterologous protein (bovine pancreatic trypsin inhibitor; BPTI) fused to the mature major coat protein (gene VIII product; VIII) of bacteriophage M13 has been demonstrated. Optimization of the promoter, signal peptide and host bacterial strain allowed for the construction of a working vector consisting of the M13 genome, into which was cloned a synthetic gene composed of a lac (or tac) promoter, and sequences encoding the bacterial alkaline phosphatase signal peptide, mature BPTI and the mature coat protein. Processing of the BPTI-VIII fusion protein and its incorporation into the bacteriophage were found to be maximal in a host bacterial strain containing a prlA/secY mutation. Functional protein is displayed on the surface of M13 phage, as judged by specific interactions with antiserum, anhydrotrypsin, and trypsin. Such display vectors can be used for epitope mapping, production of artificial vaccines and the screening of diverse libraries of proteins or peptides having affinity for a chosen ligand. The VIII display phage system has practical advantages over the III display phage system in that many more copies of the fusion protein can be displayed per phage particle and the presence of the VII fusion protein has little or no effect on the infectivity of the resulting bacteriophage.     

The harnessing of peptide–monolith constructs for single step plasmid DNA purification

The availability of synthetic peptides has paved the way for their use in tailor-made interactions with biomolecules. In this study, a 16mer LacI-based peptide was used as an affinity ligand to examine the scale up feasibility for plasmid DNA purification. First, the peptide was designed and characterized for the affinity purification of lacO containing plasmid DNA, to be employed as a high affinity ligand for the potential capturing of plasmid DNA in a single unit operation. It was found there were no discernible interactions with a control plasmid that did not encode the lacO nucleotide sequence. The dissociation equilibrium constant of the binding between the 16mer peptide and target pUC19 was 5.0 ± 0.5 × 10⁻⁸ M as assessed by surface plasmon resonance. This selectivity and moderated affinity indicate that the 16mer is suitable for the adsorption and chromatographic purification of plasmid DNA. The suitability of this peptide was then evaluated using a chromatography system with the 16mer peptide immobilized to a customized monolith to purify plasmid DNA, obtaining preferential purification of supercoiled pUC19. The results demonstrate the applicability of peptide–monolith supports to scale up the purification process for plasmid DNA using designed ligands via a biomimetic approach.     

Phage display of random peptide libraries: applications, limits, and potential

The identification of ligands from large biological libraries by phage display has now been used for almost 15 years. Most of the successful reports on high-affinity ligand identification originated from work with different antibody libraries. In contrast, the progress of applying phage display to random peptide libraries was relatively slow. However, in the last few years several improvements have led to an increasing number of published peptide ligands identified by phage display from such libraries and which exhibited good biological activity and high affinity. This review summarizes the current state and the technical progress of the application of random peptide libraries using filamentous phage for ligand identification.     

犬种布鲁氏菌ZG株单克隆抗体4H3抗原模拟表位的筛选及分析

【背景】目前犬布鲁氏菌病诊断存在一定的困难。【目的】筛选并研究犬种布鲁氏菌单克隆抗体4H3株的特异性抗原表位。【方法】利用噬菌体肽库展示技术,以犬种布鲁氏菌单克隆抗体4H3株作为靶分子,包被酶标板,用12肽随机肽库经过3轮生物淘洗程序进行筛选。经过3轮筛选后,噬菌体产出率从5.00×10^-7增加到9.84×10^-6,假阳性率逐轮降低。从第3轮筛选的阳性克隆中随机挑取14个进行增殖,提取基因组DNA,进行测序分析;并通过iELISA和cELISA检测阳性克隆的亲和性和特异性。【结果】14株阳性单克隆噬菌体共出现3种不同的短肽序列,分别是KMSIRHPIRLPI、ILRRRRKRIIQI和QRIHMRLTTQS;iELISA结果表明3种短肽序列与单克隆抗体的亲和性依次为KMSIRHPIRLPI>ILRRRRKRIIQI>QRIHMRLTTQS;cELISA结果显示短肽KMSIRHPIRLPI和ILRRRRKRIIQI特异性较强。对亲和性较强、特异性较高的2条短肽KMSIRHPIRLPI和ILRRRRKRIIQI展开具体分析,比对分析表...     

Combinatorial de novo design and application of a biomimetic affinity ligand for the purification of human anti‐HIV mAb 4E10 from transgenic tobacco

Monoclonal anti‐HIV antibody 4E10 (mAb 4E10) is one of the most broadly neutralizing antibodies against HIV, directed against a specific epitope on envelope protein gp41. In the present study, a combinatorial de novo design approach was used for the development of a biomimetic ligand for the affinity purification of mAb 4E10 from tobacco transgenic extract in a single chromatographic step. The biomimetic ligand (4E10lig) was based on a L ‐Phe/β‐Ala bi‐substituted 1,3,5‐triazine (Trz) scaffold (β‐Ala‐Trz‐L ‐Phe, 4E10lig) which potentially mimics the more pronounced electrostatic and hydrophobic interactions of mAb 4E10‐binding sequence determined by screening of a random peptide library. This library was comprised of Escherichia coli cells harboring a plasmid (pFlitrx) engineered to express a fusion protein containing random dodecapeptides that were inserted into the active loop of thioredoxin, which itself was inserted into the dispensable region of the flagellin gene. Adsorption equilibrium studies with this biomimetic ligand and mAb 4E10 determined a dissociation constant (K_D) of 0.41 ± 0.05 µM. Molecular modeling studies of the biomimetic ligand revealed that it can potentially occupy the same binding site as the natural binding core peptide epitope. The biomimetic affinity adsorbent was exploited in the development of a facile mAb 4E10 purification protocol, affording mAb 4E10 of high purity (approximately 95%) with good overall yield (60–80%). Analysis of the antibody preparation by SDS‐PAGE, enzyme‐linked immunosorbent assays (ELISA), and western blot showed that the mAb 4E10 was fully active and free of degraded variants, polyphenols, and alkaloids. Copyright © 2009 John Wiley & Sons, Ltd.     

Metal chelate affinity precipitation: a new approach to protein purification

Metal chelate affinity precipitation of proteins, a method combining metal–protein interaction and affinity precipitation is being discussed as a selective separation process for proteins. The technique utilizes a flexible soluble–insoluble thermo-responsive polymer with a covalently linked ligand loaded with metal ions. The affinity binding of the target protein varies with different metal ions. Copolymers of N-isopropylacrylamide with 1-vinylimidazole loaded with Cu(II) ions are designed as a potential carriers for affinity purification and proved to be successful for purification of protein inhibitors from a variety of cereals. This revised version was published online in July 2006 with corrections to the Cover Date.     

Microscale affinity purification of trypsin reduces background peptides in matrix-assisted laser desorption/ionization mass spectrometry of protein digests

The use of trypsin for protein digestion is hampered by its autolysis and low thermostability. Chemical modifications have been employed to stabilize the enzyme. Modified trypsin (e.g. methylated) usually enables performing digestions at elevated temperatures, but it still produces autolytic peptides. In this work, unmodified bovine trypsin was subjected to a microscale affinity chromatography on Arginine Sepharose (ASE) or Benzamidine Sepharose (BSE), which utilized the principle of active-site ligand binding. Trypsin was retained on the sorbents in ammonium bicarbonate as a binding buffer. After washings to remove unbound impurities, the enzyme was eluted by arginine as a free ligand (from ASE) or by diluted hydrochloric acid (from BSE). MALDI-TOF mass spectrometry confirmed removal of large molecular fragments as well as autolytic and other background peptides. Consequently, the purified trypsin was tested for its performance in procedures of in-gel digestion of protein standards and selected urinary proteins from real samples. It has been shown that the affinity purification of trypsin decreases significantly the number of unmatched peptides in peptide mass fingerprints. The presence of arginine in the digestion buffer was found to reduce intensity of autolytic peptides. As a result, the described purification procedure is applicable in a common proteomic routine.