Recombinant proteins L and LG

Several bacterial cell wall proteins, like proteins A and G, with valuable affinity for immunoglobulins have been discovered and are currently employed in immunological techniques. In 1988, protein L, a bacterial cell wall protein with Ig-binding capacity, was isolated from the anaerobic bacterial speciesPeptostreptococcus magnus. Binding data with immunoglobulin fragments suggested that protein L could selectively bind the variable region of human kappa light chains. More recently a recombinant LG fusion protein was expressed inE. coli containing four repeated Ig-binding domains of protein L (fragment B1–4) and two IgG Fc-binding protein G domains (fragment CDC). Recombinant L and LG proteins were tested in the purification of murine monoclonal IgG and their fragments. After affinity-constant evaluation in different buffer systems, high-pressure affinity-chromatography columns were prepared by coupling the proteins to Affi-prep 10 resin and tested with eight different murine monoclonal antibodies and their fragments of various isotypes. Affinity-chromatography experiments confirming radioimmunoassay results showed 75% fragment-binding capacity of protein L and 100% of protein LG. These results evidenced protein LG as the most powerful Ig-binding tool so far described. Therefore, application of these proteins may be suggested in the purification of murine immunoglobulins and their fragments, including the engineered ones.     

Protein LG: a hybrid molecule with unique immunoglobulin binding properties.

Immunoglobulin (Ig)-binding bacterial proteins have attracted theoretical interest for their role in molecular host-parasite interactions, and they are widely used as tools in immunology, biochemistry, medicine, and biotechnology. Protein L of the anaerobic bacterial species Peptostreptococcus magnus binds Ig light chains, whereas streptococcal protein G has affinity for the constant (Fc) region of IgG. In this report, Ig binding parts of protein L and protein G were combined to form a hybrid molecule, protein LG, which was found to bind a large majority of intact human Igs as well as Fc and Fab fragments, and Ig light chains. Binding to Ig was specific, and the affinity constants of the reactions between protein LG and human IgG, IgGFc fragments, and kappa light chains, determined by Scatchard plots, were 5.9 x 10(9), 2.2 x 10(9), and 2.0 x 10(9) M-1, respectively. The binding properties of protein LG were more complete as compared with previously described Ig-binding proteins when also tested against mouse and rat Igs. This hybrid protein thus represents a powerful tool for the binding, detection, and purification of antibodies and antibody fragments.     

Cathepsin L Causes Proteolytic Cleavage of Chinese-Hamster-Ovary Cell Expressed Proteins During Processing and Storage: Identification,Characterization, and Mitigation

A stochastic approach of copurification of the protease Cathepsin L that results in product fragmentation during purification processing and storage is presented. Cathepsin L was identified using mass spectroscopy, characterization of proteolytic activity, and comparison with fragmentation patterns observed using recombinant Cathepsin L. Cathepsin L existed in Chinese hamster ovary cell culture fluids obtained from cell lines expressing different products and cleaved a variety of recombinant proteins including monoclonal antibodies, antibody fragments, bispecific antibodies, and fusion proteins. Therefore, characterization its chromatographic behavior is essential to ensure robust manufacturing and sufficient shelf life. The chromatographic behaviors of Cathepsin L using a variety of techniques including affinity, cation exchange, anion exchange, and mixed mode chromatography were systematically evaluated. Our data demonstrates that copurification of Cathepsin L on nonaffinity modalities is principally because of similar retention on the stationary phase and not through interactions with product. Lastly, Cathespin L exhibits a broad elution profile in cation exchange chromatography (CEX) likely because of its different forms. Affinity purification is free of fragmentation issue, making affinity capture the best mitigation of Cathepsin L. When affinity purification is not feasible, a high pH wash on CEX can effectively remove Cathepsin L but resulted in significant product loss, while anion exchange chromatography operated in flow-through mode does not efficiently remove Cathepsin L. Mixed mode chromatography, using Capto™ adhere in this example, provides robust clearance over wide process parameter range (pH 7.7 ± 0.3 and 100 ± 50 mM NaCl), making it an ideal technique to clear Cathepsin L. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2732, 2019     

Using precipitation by polyamines as an alternative to chromatographic separation in antibody purification processes

Polyamine precipitation conditions for removing host cell protein impurities from the cell culture fluid containing monoclonal antibody were studied. We examined the impact of polyamine concentration, size, structure, cell culture fluid pH and ionic strength. A 96-well microtiter plate based high throughput screening method was developed and used for evaluating different polyamines. Polyallylamine, polyvinylamine, branched polyethyleneimine and poly(dimethylamine-co-epichlorohydrin-ethylenediamine) were identified as efficient precipitants in removing host cell protein impurities. Leveraging from the screening results, we incorporated a polyamine precipitation step into a monoclonal antibody purification process to replace the Protein A chromatography step. The optimization of the overall purification process was performed by taking the mechanisms of both precipitation and chromatographic separation into account. The precipitation-containing process removed a similar amount of process-related impurities, including host cell proteins, DNA, insulin and gentamicin and maintained similar product quality in respect of size and charge variants to chromatography based purification. Overall recovery yield was comparable to the typical Protein A affinity chromatography based antibody purification process.     

Separation of histones from Physarum polycephalum by ion-paired, reverse-phase high-performance liquid chromatography

A rapid, one-step method for the efficient purification of murine monoclonal antibodies from tissue culture supernatants is described. This process is based on affinity chromatography on protein A-Sepharose columns. It was found that murine monoclonal antibodies raised against tick-borne encephalitis virus frequently eluted at more than one pH value and these pH values did not always correspond to those of antibodies of the same subclass from polyclonal mouse sera. The two populations of antibody molecule eluting at different pH values showed no variation in molecular weight, isoelectric profiles, specific enzyme-linked immunosorbent assay titer, or antibody subclass.     

Temperature-triggered purification of antibodies

In this article the unique capability of elastin-like protein (ELP) to reversibly precipitate was combined with the high affinity and specificity of antibody-binding domains such as Protein G, Protein L, or Protein LG as a general method for antibody purification that combines in a unique manner the simplicity and robustness of temperature-triggered precipitation with the selectivity of affinity interactions. In a single precipitation step, antibodies derived from different sources (animal sera or hybridoma cell cultures) were selectively recovered by a simple temperature trigger. Due to the versatility of the binding ligands toward different classes of antibodies, we believe that this technology will be useful as an economical, highly efficient, and universal platform for the purification of antibodies.     

Glycoprotein gp118 of varicella-zoster virus: purification by serial affinity chromatography.

Glycoprotein gp118, one of the major glycosylated proteins specified by varicella-zoster virus, is biologically of great importance since it possesses an epitope which elicits a complement-independent neutralizing antibody response. To purify this glycoprotein from a Nonidet-solubilized extract of varicella-zoster virus-infected cells, we examined its affinity to a variety of ligands, including two lectins--concanavalin A and Lens culinaris, Cibacron blue and heparin, and finally an immunoadsorbent anti-gp118 monoclonal antibody. By serial affinity chromatography on three different columns consisting of, respectively (i) Cibacron blue dye-Sepharose, (ii) L. culinaris-Sepharose, and (iii) anti-gp118 murine monoclonal antibody bound to CNBr-activated Sepharose, we isolated varicella-zoster virus-specific gp118 essentially free of contamination by any other radiolabeled viral or cellular polypeptide. The fold purification was estimated at 1,025 and the percent recovery at 13.6. On the basis of its chromatographic properties, gp118 appeared to contain mainly asparagine-linked, biantennary, complex-type, and hybrid-type oligosaccharides.     

Purification of Neuronal Cell Surface Proteins and Generation of Epitope-Specific Monoclonal Antibodies Against Cell Adhesion Molecules

To establish a procedure for the purification of a broad spectrum of cell surface proteins, three separate methods based on different principles were compared with the aid of four marker proteins. Membrane preparation by sedimentation-flotation centrifugation, temperature-induced phase separation with Triton X-114, and lectin affinity chromatography were used separately as well as in combination. The two-step procedure of membrane preparation and lectin affinity chromatography provided by far the best enrichment of cell surface marker proteins. This result was further substantiated by screening greater than 6,600 hybridoma cultures that originated from mice that had been immunized with protein fractions obtained by different purification protocols. In addition, it was found that solubilized glycoproteins used as immunogens led to many more cell surface-specific monoclonal antibodies than glycoproteins immobilized on lectin-agarose beads. Three monoclonal antibodies that recognize distinct epitopes of cell adhesion molecules (CAMs) were isolated. Monoclonal antibody C4 bound to a detergent-labile epitope of G4 (neuron-glia CAM). Monoclonal antibody D1 recognized specifically nonreduced neural CAM (N-CAM) with intact disulfide bridges, and monoclonal antibody D3 recognized only the 180-kilodalton isoform of N-CAM. Because of these specificities, these monoclonal antibodies promise to be useful tools for the elucidation of the structural organization of adhesion molecules.     

Purification and characterization of an erythrocyte membrane protein complex carrying Duffy blood group antigenicity. Possible receptor for Plasmodium vivax and Plasmodium knowlesi malaria parasite

A murine monoclonal antibody, named anti-Fy6, which agglutinates all human red cells except those of Fy(a-b) phenotype was used for purification and characterization of Duffy antigens. Duffy antigens are multimeric red cell membrane proteins composed of different subunits of which only one, designated pD protein, reacts in immunoblots with the murine monoclonal antibody anti-Fy6. Affinity-purified detergent-soluble antigen-antibody complex obtained from red cells, surface-labeled with 125I yielded a complex pattern of bands when separated by polyacrylamide gel electrophoresis. Proteins that react with anti-Fy6 in immunoblots are: pA and pB (greater than 100 kDa) and pD (36-46 kDa). Electroeluted pD protein aggregates and generates bands of similar molecular mass to pA and pB proteins. Electroeluted pA and pB proteins disaggregate yielding pD protein. Oligomers and monomers of pD protein are present in red cells carrying Duffy antigens and absent in Fy(a-b-) cells. Six other proteins of molecular weight ranging from 68 to 21 kDa either associate or co-purify with pD protein. These proteins are only present in Duffy antigen positive cells. The pD protein is different in Fy(a+b-) and Fy(a-b+) cells by fingerprint analysis. Human antisera identify the same proteins in red cell carrying Duffy antigens as the murine monoclonal antibody anti-Fy6.     

A strategy to identify linker-based modules for the allosteric regulation of antibody-antigen binding affinities of different scFvs

Antibody single-chain variable fragments (scFvs) are used in a variety of applications, such as for research, diagnosis and therapy. Essential for these applications is the extraordinary specificity, selectivity and affinity of antibody paratopes, which can also be used for efficient protein purification. However, this use is hampered by the high affinity for the protein to be purified because harsh elution conditions, which may impair folding, integrity or viability of the eluted biomaterials, are typically required. In this study, we developed a strategy to obtain structural elements that provide allosteric modulation of the affinities of different antibody scFvs for their antigen. To identify suitable allosteric modules, a complete set of cyclic permutations of calmodulin variants was generated and tested for modulation of the affinity when substituting the linker between VH and VL. Modulation of affinity induced by addition of different calmodulin-binding peptides at physiologic conditions was demonstrated for 5 of 6 tested scFvs of different specificities and antigens ranging from cell surface proteins to haptens. In addition, a variety of different modulator peptides were tested. Different structural solutions were found in respect of the optimal calmodulin permutation, the optimal peptide and the allosteric effect for scFvs binding to different antigen structures. Significantly, effective linker modules were identified for scFvs with both VH-VL and VL-VH architecture. The results suggest that this approach may offer a rapid, paratope-independent strategy to provide allosteric regulation of affinity for many other antibody scFvs.     

Synthesis and expression in Escherichia coli of DNA encoding the murine lambda 1 chain of a monoclonal antibody specific for Salmonella serotype B O-antigen.

A 658 bp DNA sequence corresponding to the murine lambda 1 chain of a monoclonal antibody, Se155-4, specific for the Salmonella serotype B O-antigen, was designed using Escherichia coli preferred codons and chemically synthesized by ligation of synthetic fragments into a linearized plasmid followed by transformation into E. coli. A synthetic signal peptide (ompA) was fused to express the L chain as a free polypeptide into the periplasm of E. coli cells. After isolation and purification, heterologous recombination of the E. coli L chain with mouse H chain gave an active antigen-binding protein. The activity was 15-20% when compared to protein created by an equivalent association of isolated natural mouse L and H chains as measured by a direct EIA assay. In inhibition experiments with the polysaccharide antigen, the two proteins showed identical titration curves and 50% inhibition points, indicating comparable KA values.     

Challenges and recent advances in affinity purification of tag-free proteins

There is currently no generic, simple, low-cost method for affinity chromatographic purification of proteins in which the purified product is free of appended tags. Existing approaches for the purification of tagless proteins fall into two broad categories: (1) direct affinity-based capture of tag-free proteins that utilize affinity ligands specific to the target protein or class of target protein, and (2) removal of an appended affinity tag following tag-mediated protein capture. This paper reviews current state-of-the-art approaches for tagless protein purification in both categories, including specific examples of affinity ligands used for the capture of different classes of proteins and cleavage systems for affinity tag removal following chromatographic capture. A particular focus of this review is on recent developments in affinity tag removal systems utilizing split inteins.     

Analysis of C3-receptor activity on human B-lymphocytes and isolation of the complement receptor type 2 (CR2).

The rosetting of defined C3-fragment-coated sheep erythrocytes to B-cell-enriched tonsil lymphocytes was measured. The rosetting lymphocytes were homogeneous with respect to expression of C3b, iC3b and C3d receptors. Isolation of receptors for C3 fragments from surface-radioiodinated lymphocytes by affinity chromatography on immobilized C3u, iC3b and C3d,g produced two proteins with partially overlapping specificities. A protein of 240 000 Mr, recognized by the monoclonal antibody To5 and identified as CR1 (complement receptor type 1), had affinity for C3u and iC3b. A protein of 145 000 Mr, recognized by the monoclonal antibody B2, had affinity for all three C3 fragments. Inhibition of rosetting by antibodies to these proteins indicates that CR1 is responsible for C3b-mediated rosetting and that the 145000-Mr receptor (CR2) is responsible for C3d-mediated rosetting. Partial inhibition by both anti-CR1 and anti-CR2 antibodies of iC3b-mediated rosetting indicates that both receptors are involved in iC3b-mediated rosetting. No other protein appears to be involved in tonsil B-cell rosetting to C3-fragment-coated cells. A method for preparing CR2 from tonsil lymphocytes based on affinity chromatography on C3d,g-Sepharose has been developed. Forty tonsil pairs (2 X 10(10) B-cells) yield about 40 micrograms of pure protein equivalent to a purification of 6500-fold from a detergent extract.     

Differential recognition of calmodulin-enzyme complexes by a conformation-specific anti-calmodulin monoclonal antibody

An anti-calmodulin monoclonal antibody having an absolute requirement for Ca2+ has been produced from mice immunized with a mixture of calmodulin and calmodulin-binding proteins. Radioimmune assays were developed for the determination of its specificity. the epitope for this antibody resides on the COOH-terminal half of the mammalian protein. Plant calmodulin or troponin C had little reactivity. The apparent affinity of the antibody for calmodulin was increased approximately 60-fold in the presence of heart calmodulin-dependent phosphodiesterase. The presence of heart phosphodiesterase in the radioimmune assay greatly enhanced the sensitivity for calmodulin. The intrinsic calmodulin subunit of phosphorylase kinase and calmodulin which was bound to brain phosphodiesterases was also recognized with high affinity by the antibody. The antibody reacted poorly with calmodulin which was bound to heart or brain calcineurin, skeletal muscle myosin light chain kinase, or other calmodulin-binding proteins. In direct binding experiments, most of the calmodulin-binding proteins studied were unreactive with the antibody. This selectivity allowed purification of heart and two brain calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes on immobilized antibody affinity columns. Phosphodiesterase activity was adsorbed directly from crude samples and specifically eluted with EGTA. Isozyme separation was accomplished using a previously described anti-heart phosphodiesterase monoclonal antibody affinity support. The brain isozymes differed not only in reactivity with the anti-phosphodiesterase antibody, but also in apparent subunit molecular weight, and relative specificity for cAMP and cGMP as substrates. The calmodulin activation constants for the brain enzymes were 10-20-fold greater than for the heart enzyme. The data suggest that the binding of ligands to Ca2+/calmodulin induce conformation changes in calmodulin which alter reactivity with the anti-calmodulin monoclonal antibody. The differential antibody reactivity toward calmodulin-enzyme complexes indicates that target proteins either induce very different conformations in calmodulin and/or interact with different geometries relative to the antibody binding site. The anti-calmodulin monoclonal antibody should be useful for the purification of other calmodulin-dependent phosphodiesterases as well as isozymes of phosphorylase kinase.     

Rapid chromatographic monitoring of bioprocesses

This paper describes the use of rapid chromatographic separation systems to monitor the level of specific proteins in various bioprocesses such as downstream processing and fermentation. In these monitoring systems, samples of the liquid are continuously extracted from the process and the proteins resolved from one another by a rapid chromatographic separation. The peak on the chromatogram corresponding to the protein of interest is identified and quantified to obtain on-line information on the level of that protein in the bioprocess. There are a number of advantages in using affinity separations as the rapid chromatographic principle. In particular, the use of immobilised monoclonal antibodies potentially allows a chromatographic sensor to be constructed for any protein against which a suitable antibody can be raised. The potential of this technique is illustrated with various examples, including measurement of the levels of monoclonal antibody in tissue culture supernatant using immobilised Protein A as the affinity adsorbent. A discussion of the inherent limitations of this type of protein biosensor is also included.     

Monoclonal antibody (VII-M31) to bovine factor VII: a specific epitope in the gamma-carboxyglutamic acid domain.

A murine monoclonal antibody (designated VII-M31) directed against bovine factor VII was prepared and characterized. Antibody VII-M31 inhibited the activations of both factors IX and X catalyzed by factor VIIa in the presence of tissue factor, phospholipids, and Ca2+. It possessed a strong affinity for factor VII in the presence of 5 mM Ca2+ (Kd = 1.12 x 10(-10)M). The immunoblotting test of other bovine proteins with the antibody, such as prothrombin, factor X, factor IX, protein C, protein S, and protein Z, in addition to human factor VII, revealed that it recognizes only a Ca2(+)-dependent epitope in bovine factor VII. Furthermore, this antibody VII-M31 covalently coupled with Affi-Gel allowed a simple and rapid purification of bovine factor VII. To localize the antigenic site in factor VII, various segments including a gamma-carboxyglutamic acid (Gla)-domainless protein, a Gla-domain peptide and the fragments isolated from the lysyl endopeptidase digest, were prepared. Among them, the isolated Gla-domain peptide and Gla-domainless factor VII were no longer recognized by antibody VII-M31, indicating that the sequence around the cleavage site by a-chymotrypsin is required for the interaction between the antibody and factor VII. In accordance with this result, the antibody bound specifically to a Gla-containing peptide corresponding to the NH2-terminal 23-50 residues of factor VII, which contains the chymotryptic cleavage site. These results suggest that the specific epitope of this antibody is localized in the carboxy-terminal 28 residues of the Gla-domain constituting the amino-terminal portion of bovine factor VII.     

Preparation and characterization of two monoclonal antibodies against different epitopes in Escherichia coli ribosomal protein L7/L12

Two monoclonal antibodies with specificities for Escherichia coli 50 S ribosomal subunit protein L7/L12 were isolated. The antibodies and Fab fragments thereof were purified by affinity chromatography using solid-phase coupled L7/L12 protein as the immunoadsorbent. The two antibodies were shown to recognize different epitopes; one in the N-terminal and the other in the C-terminal domain of protein L7/L12. Both intact antibodies strongly inhibited polyuridylic acid-directed polyphenylalanine synthesis, ribosome-dependent GTPase activity, and the binding of elongation factor EF-G to the ribosome. Ratios of antibody to ribosome of 4:1 or less were effective in inhibiting these activities. Neither antibody prevented the association of ribosomal subunits to form 70 S ribosomes. The Fab fragments showed similar effects.     

Purification of histidine-tagged single-chain Fv-antibody fragments by metal chelate affinity precipitation using thermoresponsive copolymers

Metal chelate affinity precipitation (MCAP) has been successfully developed as a simple purification process for proteins that have affinity for metal ions. The present lack of widespread applications for this technique as compared to immobilized metal affinity chromatography (IMAC) may be related to the scarcity of well-characterized metal affinity macroligands (AML) and their applications to the number of different purification systems. In the present work we describe a detailed study of a new purification system using metal-loaded thermoresponsive copolymers as AML. The copolymers of vinylimidazole (VI) with N-isopropylacrylamide (NIPAM) were synthesized by radical polymerization with imidazole contents of 15 and 24 mol%. When loaded with Cu(II) and Ni(II) ions the copolymers selectively precipitated extracellularly expressed histidine-tagged single-chain Fv-antibody fragments (His(6)-scFv fragments) from the fermentation broth free from E. coli cells. Precipitation was induced by salt at mild temperatures and the bound antibody fragments were recovered by dissolving the protein-polymer complex in EDTA buffer and subsequent reprecipitation of the polymer. His(6)-scFv fragments were purified with yields of 91 and 80% and purification folds of 16 and 21 when Cu(II) and Ni(II) copolymers were used, respectively. The protein precipitation capacity of the Ni(II) copolymer showed a dependence on the VI concentration in the copolymer. The SDS-PAGE pattern showed significant purification of the antibody fragments.     

Large-surface biosensor technology for enhanced recovery in protein characterization.

A large-surface biosensor technique using surface plasmon resonance (SPR) was tested for protein purification by recovery of a monoclonal antibody against human proinsulin C-peptide. Notably, both reversible attachment/desorption and actual purification of the antibody from a multi-component protein mixture was shown. For initial chip attachment of the peptide ligand, C-peptide was biotinylated and attached to neutravidin on plastic chips with a large gold surface (effective area 26 mm(2)). Antibody binding and desorption was monitored in real-time SPR, and for elution different conditions were employed. Five percent formic acid (in contact with the chip surface for 3 min) in a 60-mul segment between air bubbles was efficient for subsequent analysis. In this manner, protein amounts up to 35 pmoles were recovered in a single capture/elution cycle. Evaluation by SDS-PAGE showed essentially no carryover between fractions in this elution process, and also not with other proteins in the mixture after purification. Compared to existing commercial instruments, this technique gives higher recovery and makes it possible to monitor monitor protein binding/desorption. Recovery of affinity partners at the multi-pmole level is demonstrated for protein purification in SPR approaches.     

Methods for preparation of low abundance glycoproteins from mammalian cell supernatants

Proteins secreted to mammalian cell supernatants are usually in a low concentration and purity, due to the limitation of the expression systems or the presence of a large amount of contaminant proteins from the cell medium. So, initial protein recovery from cell supernatants requires of a highly specific chromatography step. We compared several purification methods based on affinity chromatography for purification of proteins from cell culture supernatants: metal chelate affinity, strep-tag and immunopurification with a monoclonal antibody. Soluble receptor glycoproteins were engineered with the corresponding peptide tag at their C-terminal end. The proteins were expressed in 293T cells and secreted to the cell supernatant, as monitored by sandwich ELISA. Supernatants were run through the different chromatography columns and several purification-related parameters determined. While all column-retained proteins were easily eluted, the chelating and immunopurification chromatography gave the highest yield and the latest method provided a sample with the highest purity. So, in spite of its cost, immunopurification chromatography gave optimal results for purification of a low abundance protein from a cell supernatant. Finally, we applied a protein expression system together with immunopurification chromatography for preparation of a glycoprotein for crystallization.