Selection of affinity ligands for protein purification from proteolytic digests

A simple method for the selection of affinity ligands from proteolytic digests by affinity chromatography is proposed. A small proportion of the peptides in the trypsin digest of bovine serum albumin (BSA) or the pepsin digest of cytochrome are retarded on insulin-immobilised or HSA (human serum albumin)-immobilised affinity columns, respectively. The peptides in these selected fractions can be immobilised onto solid phases and used in affinity chromatography procedures for the purification of insulin or HSA. © Rapid Science Ltd. 1998     

Utilizing a library of synthetic affinity ligands for the enrichment, depletion and one-step purification of leech proteins

Although the concept of affinity purification using synthetic ligands had been utilized for many years, there are few articles related to this research area, and they focus only on the affinity purification of specific protein by a defined library of synthetic ligands. This study presents the design and construction of a 700-member library of synthetic ligands in detail. We selected 297 ligand columns from a 700-member library of synthetic ligands to screen leech protein extract. Of the 297, 154 columns had an enrichment effect, 83 columns had a depletion effect, 36 columns had a one-step purification effect, and 58 columns had a one-step purification via flowthrough effect. The experimental results achieved by this large library of affinity ligands provide solid convincing data for the theory that affinity chromatography could be used for the enrichment of proteins that are present in low abundance, the depletion of high abundance proteins, and one-step purification of special proteins.     

Identification of affinity ligands for protein purification from synthetic chemical combinatorial libraries

A method to screen combinatorial libraries for the development of selective ligands for protein affinity chromatographic purification is described. The method is based on the application of parallel combinatorial libraries, and it has several potential advantages. The screening procedure is simple and straightforward, and it does not require the chemical derivatization of the target proteins or even that the target protein be pure. The experiment can also be designed to select binders that are less likely to cause protein denaturation. Feasibility of this approach is demonstrated with a model study of the chromatographic purification of bovine albumin serum (BSA) and Avidin.     

Monoclonal antibody purification by affinity chromatography with ligands derived from the screening of peptide combinatory libraries

The peptide, Ala-Pro-Ala-Arg (APAR), was selected from the screening of a tetrapeptide combinatorial synthetic library as the ligand for affinity purification of an anti-Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) monoclonal antibody (Mab) developed in mouse ascitis. The affinity chromatographic matrix obtained by attachment of APAR to agarose, having a peptide density of 0.5 mol ml^–1, showed a maximum capacity of 9.1 mg Mab ml^–1 and a dynamic capacity of 3.9 mg Mab ml^–1. A 95% yield of electrophoretically pure anti-GM-CSF was obtained in a single step.     

Study on the scale-up of human IgG3 purification using protein A affinity chromatography

The purification of human IgG3 subclass out of IgG (Immunoglobulin-G) was studied using protein A-Sepharose affinity chromatography. The effect of operational parameters such as flow rate, ionic strength, pH and size of sample was investigated, and the process was scaled-up 10-fold. The use of 0.5 m NaCl in the loading buffer had a dramatic effect in the purity of IgG3 recovered in the flowthrough fraction (values in the order of 97% were consistently obtained). This was attributed to a more effective binding of IgG subclasses 1, 2 and 4 to protein A (well known classical mechanism based in Fc fragment) and in some extent to a decrease in the binding of subclass 3 to protein A by the alternative mechanism based in the Fab fragment. The increase in residence time also increased in a relevant way the purity of IgG3. This is attributed to an increased effectiveness of the mechanisms mentioned above. The recovery yields in the IgG3 rich fraction were in the range 21-32% and are possibly a consequence of binding to protein A by the alternative mechanism and also due to deactivation during processing.     

Affinity cross-flow filtration: purification of IgG with a novel protein a affinity matrix prepared from two-dimensional protein crystals

In this article, we describe the use of 1- to 2-mum sized affinity microparticles for the isolation and purification of IgG from artificial IgG-human serum albumin mixtures and clarified hybridoma cell culture supernatants by affinity cross-flow filtration. Affinity microparticles were prepared from cell wall fragments of Clostridium thermohydrosulfuricum L111-69, in which the peptidoglycan-containing layer was completely covered with a hexagonally ordered S-layer lattice. After crosslinking the S-layer protein with glutaraldehyde, carboxyl groups from acidic amino acids were activated with carbodiimide and used for immobilization of Protein. A. Quantitative determination confirmed that Protein A molecules formed a monomolecular layer on the outermost surface of the S-layer lattice. Affinity microparticles were found to withstand high centrifugal and shear forces and revealed no Protein A leakage or S-layer protein release under cross-flow conditions between pH 2 to 12. The IgG-binding capacity of affinity microparticles was investigated under crossflow conditions and compared with that obtained in batch adsorption processes. (c) 1994 John Wiley & Sons, Inc.     

G蛋白亲和色谱法纯化动物血清、抗体效果比较

比较 Hitrap G蛋白 Sepharose亲和色谱系统对不同动物血清或腹水的纯化效果 ,为抗体纯化提供依据。结果显示 ,G蛋白对不同动物血清 Ig G吸附能力不同 ,体现在单位体积抗体回收量明显不同 ,这一特点与 A蛋白亲和色谱系统相似。该方法简便快速 ,纯化抗体纯度高 ,免疫活性好 ,色谱柱可反复使用多次     

Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element

A novel protein purification system has been developed which enables purification of free recombinant proteins in a single chromatographic step. The system utilizes a modified protein splicing element (intein) from Saccharomyces cerevisiae (Sce VMA intein) in conjunction with a chitin-binding domain (CBD) from Bacillus circulans as an affinity tag. The concept is based on the observation that the modified Sce VMA intein can be induced to undergo a self-cleavage reaction at its N-terminal peptide linkage by 1,4-dithiothreitol (DTT), β-mercaptoethanol (β-ME) or cysteine at low temperatures and over a broad pH range. A target protein is cloned in-frame with the N-terminus of the intein-CBD fusion, and the stable fusion protein is purified by adsorption onto a chitin column. The immobilized fusion protein is then induced to undergo self-cleavage under mild conditions, resulting in the release of the target protein while the intein-CBD fusion remains bound to the column. No exogenous proteolytic cleavage is needed. Furthermore, using this procedure, the purified free target protein can be specifically labeled at its C-terminus.     

Synthesis and screening of a rationally designed combinatorial library of affinity ligands mimicking protein L from Peptostreptococcus magnus

Rational design and combinatorial chemistry were utilized to search for lead protein L (PpL) mimetics for application as affinity ligands for the purification of antibodies and small fragments, such as Fab and scFv, and as potential diagnostic or therapeutic agents. Inspection of the key structural features of the complex between PpL and human Fab prompted the de novo design and combinatorial synthesis of a 169-membered solid-phase ligand library, which was assessed for binding to human IgG and subsequent selectivity for the Fab fragment. Eight ligands were selected, chemically characterized and compared with a commercial PpL-adsorbent for binding pure immunoglobulin fractions. The most promising lead, ligand 8/7, when immobilized on an agarose support, behaved in a similar fashion to PpL in isolating Fab fragments from papain digests of human IgG to a final purity of 97%.     

Non-specific depurination activity of saporin-S6, a ribosome-inactivating protein, under acidic conditions

Among five ribosome-inactivating proteins tested only saporin-S6 could efficiently release the adenine from adenosine 20 of the synthetic oligoribonucleotide (SRD RNA) mimic of the sarcin/ricin domain of rat 28S rRNA with a Km of 9 microM and a kcat of approximately 0.4 min(-1) at pH 7.6. The optimal pH for the depurination activity of saporin-S6 is 5.0. However, saporin-S6 lost its site-specificity of depurination on SRD RNA around the optimal pH. The non-specific depurination activity of saporin-S6 was dependent on the enzyme concentration and pH conditions. These results are valuable to understand the diversity and the depurination mechanism of ribosome-inactivating proteins.     

SPR identification of mild elution conditions for affinity purification of E6 oncoprotein, using a multivariate experimental design

The purification of "difficult" proteins for structural and functional studies remains a challenge. A widely used approach is their production as fusions with an affinity tag, so that a generic tag-based purification protocol can be applied. Alternatively, immuno-affinity using a protein-specific antibody allows purification of unmodified proteins in a single step, if mild elution conditions can be identified for dissociating the complex without disrupting the folding of the protein. Here, we describe a quantitative structure activity relationship (QSAR) strategy to predict optimized elution conditions from a mathematical model that relates target/antibody dissociation to environmental changes. We illustrate the strategy with the E6 protein of the human papilloma virus (HPV) 16, a highly unstable protein central to HPV-induced carcinogenesis. Surface plasmon resonance (SPR) was used to measure the kinetics of dissociation of an E6 peptide from an E6-specific antibody in a set of multivariate conditions, where three environmental factors (pH, NaCl concentration, and temperature) were varied. The QSAR model indicated that dissociation is favored at pH < 5, which is detrimental to E6 folding, and also at pH > or = 10 if the temperature is high. We verified that the conclusions of the QSAR study with the peptide were valid for the scFv1F4/E6 protein complex, and that the recovered protein was capable of mediating p53 degradation. Finally, we demonstrated that the optimized elution conditions (pH 10, 35 degrees C) were adequate for purifying the recombinant E6 protein from crude cell extracts.     

Affinity purification of proteins using ligands derived from peptide libraries

Peptide libraries can be used to identify ligands that bind specifically to a desired protein. These peptides may have significant advantages as specific ligands for affinity chromatography separations. This article describes the use of one of such peptide, Try-Asn-Phe-Glu-Val-Leu, as a ligand for the purification of S-protein using affinity chromatography. General strategies for peptide immobilization are discussed and the conditions for peptide immobilization to Emphazetrade mark gel are optimized. The effects of peptide orientation and peptide densities on protein binding are studied. Results indicate that the peptide affinity is not affected by the orientation of the peptide during immobilization, but association constants can be reduced by one order of magnitude when compared with the values in solution.With increased peptide density, the protein binding capacity of the gel increases, but both the percentage of peptide utilization and apparent binding constant between immobilized peptide and S-protein decrease. S-protein is separated from a mixture with BSA via affinity chromatography using specific elution with the peptide in solution.Finally, direct purification of S-protein from an enzymatic digestion mixture of ribonuclease A is demonstrated.(c) 1995 John Wiley & Sons, Inc.     

Biomimetic design of affinity peptide ligands for human IgG based on protein A-IgG complex

Staphylococcal protein A (SpA) has been widely used as an affinity ligand for the purification of immunoglobulin G (IgG). Based on the affinity motif of SpA, we have herein developed a biomimetic design strategy for affinity peptide ligands of IgG. First, according to the distribution of the six hot spots of the SpA affinity motif determined previously, the number of residues that should be inserted into between the hot spots was determined. Cysteine was introduced as one of the middle inserted residues of the peptide for later immobilization. Then, amino acid location was performed to identify other amino acid residues for insertion, leading to the construction of a peptide library. The library was screened by using different molecular simulation protocols, resulting in the selection of 15 peptide candidates. Thereafter, molecular dynamics simulations were performed to validate the dynamics of the affinity interactions between the candidates and IgG, and 14 of them were found to keep high affinities. Finally, the affinity and specificity of the top one ligand FYWHCLDE were exemplified by protein chromatography and IgG purification. The results indicate that the design strategy was successful and the affinity peptide ligand for IgG is promising for application in antibody purifications.     

A蛋白亲和色谱法纯化动物血清抗体的效果比较

比较Hitrap A蛋白琼脂糖凝胶亲和色谱系统,对不同动物血清或抗血清抗体的纯化效率,提供选择抗体纯化方法的依据。该方法简使快速,纯化的抗体纯度高,能较好地保持抗体免疫学活性。色谱柱反复使用40多次,纯化抗体效率不变。但A蛋白对不同动物血清的IgG吸附能力不同,对兔和豚鼠的血清抗体吸附能力强,而对小鼠、山羊和驴的血清抗体吸附能力较弱。说明虽然该方法纯化的抗体纯度高,但它对动物品种有选择。因此,应根据动物品种,选择适合的纯化方法。     

A蛋白亲和层析法纯化动物血清抗体的效果比较

用ＨｉｔｒａｐＰｒｏｔｅｉｎ－ＡＳｅｐｈａｒｏｓｅ亲和层析系统,对不同动物血清或抗血清抗体的纯化效率进行了比较,为选择抗体纯化方法提供依据。该方法简便快速,纯化的抗体纯度高,能较好地保持抗体免疫学活性。层析柱反复使用４０多次,纯化抗体效率不变。但Ｐｒｏｔｅｉｎ－Ａ对不同动物血清的ＩｇＧ吸附能力不同：对兔和豚鼠的血清抗体吸附能力强     

The ARiBo tag: a reliable tool for affinity purification of RNAs under native conditions

Although RNA-based biological processes and therapeutics have gained increasing interest, purification of in vitro transcribed RNA generally relies on gel-based methods that are time-consuming, tedious and denature the RNA. Here, we present a reliable procedure for affinity batch purification of RNA, which exploits the high-affinity interaction between the boxB RNA and the N-peptide from bacteriophage λ. The RNA of interest is synthesized with an ARiBo tag, which consists of an activatable ribozyme (the glmS ribozyme) and the λBoxB RNA. This ARiBo-fusion RNA is initially captured on Glutathione-Sepharose resin via a GST/λN-fusion protein, and the RNA of interest is subsequently eluted by ribozyme self-cleavage using glucosamine-6-phosphate. Several GST/λN-fusion proteins and ARiBo tags were tested to optimize RNA yield and purity. The optimized procedure enables one to quickly obtain (3 h) highly pure RNA (>99%) under native conditions and with yields comparable to standard denaturing gel-based protocols. It is widely applicable to a variety of RNAs, including riboswitches, ribozymes and microRNAs. In addition, it can be easily adapted to a wide range of applications that require RNA purification and/or immobilization, including isolation of RNA-associated complexes from living cells and high-throughput applications.