One-Step Purification of Recombinant Cutinase from an E. coli Extract Using a Stabilizing Triazine-Scaffolded Synthetic Affinity Ligand.

Cutinase from Fusarium solani pisi is an enzyme that bridges functional properties between lipases and esterases, with applications in detergents, food processing, and the synthesis of fine chemicals. The purification procedure of recombinant cutinase from E. coil extracts is a well-established but time-consuming process, which involves a sequence of two anionic exchange chromatography steps followed by dialysis. Affinity chromatography is the most efficient method for protein purification, the major limitation of its use being often the availability of a ligand selective for a given target protein. Synthetic affinity ligands that specifically recognize certain sites on the surface of proteins are highly desirable for affinity processes due to their cost-effectiveness, durability, and reusability across multiple cycles. Additionally, these ligands establish moderate affinity interactions with the target protein, making it possible to purify proteins under gentle conditions while maintaining high levels of activity recovery. This study aimed to develop a new method for purifying cutinase, utilizing triazine-scaffolded biomimetic affinity ligands. These ligands were previously screened from a biased-combinatorial library to ensure their binding ability to cutinase without compromising its biological function. A lead ligand, designated as 11/3', [4-({4-chloro-6-[(2-methylbutyl)amino]-1,3,5-triazin-2-yl}amino)benzoic acid], was chosen and directly synthesized onto agarose. Experiments conducted at different scales demonstrated that this ligand (with an affinity constant Ka ≈ 104 M-1) exhibited selectivity towards cutinase, enabling the purification of the enzyme from an E. coli crude production medium in a single step. Under optimized conditions, the protein and activity yields reached 25% and 90%, respectively, with a resulting cutinase purity of 85%.

Biomimetic Affinity Ligands for Protein Purification.

The development of sophisticated molecular modeling software and new bioinformatic tools, as well as the emergence of data banks containing detailed information about a huge number of proteins, enabled the de novo intelligent design of synthetic affinity ligands. Such synthetic compounds can be tailored to mimic natural biological recognition motifs or to interact with key surface-exposed residues on target proteins, and are designated as "biomimetic ligands". A well-established methodology for generating biomimetic or synthetic affinity ligands integrates rational design with combinatorial solid-phase synthesis and screening, using the triazine scaffold and analogs of amino acid side chains to create molecular diversity.Triazine-based synthetic ligands are nontoxic, low-cost, and highly stable compounds that can replace advantageously natural biological ligands in the purification of proteins by affinity-based methodologies.

Production and Purification of Therapeutic Enzymes.

The use of therapeutic enzymes embraces currently a vast array of applications, abridging from diggestive disorders to cancer therapy, cardiovascular and lysosomal storage diseases. Enzyme drugs bind and act on their targets with great affinity and specificity, converting substrates to desired products in a reduced time frame with minimal side reactions. These characteristics have resulted in the development of a multitude of enzyme biopharmaceuticals for a wide range of human disorders.The advances in genetic engineering and DNA recombination techniques facilitated the production of therapeutical human-like enzymes, using different cells as host organisms. The selection of hosts generally privileges those that secrete the enzyme into the culture medium, as this eases the purification process, and those that are able to express complex glycoproteins, with glycosylation patterns and other post-translational modifications close to human proteins. Moreover, engineering approaches such as pegylation, encapsulation in micro- and nanocarriers, and mutation of amino acid residues of the native enzyme molecule to yield variants with improved therapeutic activity, half-life and/or stability, have been also addressed. Engineered enzyme products have been designed to display enhanced delivery to target sites and reduced adverse side-effects (e.g., immunogenicity) upon continuous drug administration.Irrespectively of the production method, the final formulation of therapeutic enzymes must display high purity and specificity, and they are often marketed as lyophilized pure preparations with biocompatible buffering salts and diluents to prepare the reconstituted aqueous solution before treatment.

Biomimetic affinity ligands for protein purification.

The development of sophisticated molecular modeling software and new bioinformatic tools, as well as the emergence of data banks containing detailed information about a huge number of proteins, enabled the de novo intelligent design of synthetic affinity ligands. Such synthetic compounds can be tailored to mimic natural biological recognition motifs or to interact with key surface-exposed residues on target proteins and are designated as "biomimetic ligands." A well-established methodology for generating biomimetic or synthetic affinity ligands integrates rational design with combinatorial solid-phase synthesis and screening, using the triazine scaffold and analogues of amino acids side chains to create molecular diversity.Triazine-based synthetic ligands are nontoxic, low-cost, highly stable compounds that can replace advantageously natural biological ligands in the purification of proteins by affinity-based methodologies.

Synthesis and characterization of biomimetic nanogels for immunorecognition.

Biomimetic nanoparticles are promising materials for biomedical and biotechnological applications. Cationic poly(N-isopropylacrylamide) (PNIPAM) nanogels containing charged amine groups brought by addition of 2-aminoethylmethacrylate hydrochloride (AEMH) or N-(3-aminopropyl) methacrylamide hydrochloride (APMH) as co-monomers were prepared by surfactant-free precipitation polymerization. The influence of the relative amount and mode of addition of the co-monomer on both the size and the amine group density of the nanogel particles was studied. Two nanogels, one prepared using APMH (1%mol/mol NIPAM, in batch) and another with AEMH (2%mol/mol NIPAM, by shot addition) as co-monomers, were selected for the covalent coupling of a Protein L-mimic ligand to free amine groups on the particles. The ability of the synthesized biomimetic nanoparticles for recognizing and binding human IgG (hIgG) molecules was assessed and the selectivity toward immunoglobulin molecules evaluated.

Immunoassays: biological tools for high throughput screening and characterisation of combinatorial libraries.

In the demanding field of proteomics, there is an urgent need for affinity-catcher molecules to implement effective and high throughput methods for analysing the human proteome or parts of it. Antibodies have an essential role in this endeavour, and selection, isolation and characterisation of specific antibodies represent a key issue to meet success. Alternatively, it is expected that new, well-characterised affinity reagents generated in rapid and cost-effective manners will also be used to facilitate the deciphering of the function, location and interactions of the high number of encoded protein products. Combinatorial approaches combined with high throughput screening (HTS) technologies have become essential for the generation and identification of robust affinity reagents from biological combinatorial libraries and the lead discovery of active/mimic molecules in large chemical libraries. Phage and yeast display provide the means for engineering a multitude of antibody-like molecules against any desired antigen. The construction of peptide libraries is commonly used for the identification and characterisation of ligand-receptor specific interactions, and the search for novel ligands for protein purification. Further improvement of chemical and biological resistance of affinity ligands encouraged the "intelligent" design and synthesis of chemical libraries of low-molecular-weight bio-inspired mimic compounds. No matter what the ligand source, selection and characterisation of leads is a most relevant task. Immunological assays, in microtiter plates, biosensors or microarrays, are a biological tool of inestimable value for the iterative screening of combinatorial ligand libraries for tailored specificities, and improved affinities. Particularly, enzyme-linked immunosorbent assays are frequently the method of choice in a large number of screening strategies, for both biological and chemical libraries.

Affinity-based methodologies and ligands for antibody purification: advances and perspectives.

Many successful, recent therapies for life-threatening diseases such as cancer and rheumatoid arthritis are based on the recognition between native or genetically engineered antibodies and cell-surface receptors. Although naturally produced by the immune system, the need for antibodies with unique specificities and designed for single application, has encouraged the search for novel antibody purification strategies. The availability of these products to the end-consumer is strictly related to manufacture costs, particularly those attributed to downstream processing. Over the last decades, academia and industry have developed different types of interactions and separation techniques for antibody purification, affinity-based strategies being the most common and efficient methodologies. The affinity ligands utilized range from biological to synthetic designed molecules with enhanced resistance and stability. Despite the successes achieved, the purification "paradigm" still moves interests and efforts in the continuous demand for improved separation performances. This review will focus on recent advances and perspectives in antibody purification by affinity interactions using different techniques, with particular emphasis on affinity chromatography.

Adsorption of human IgG on to poly(N-isopropylacrylamide)-based polymer particles.

Thermosensitive poly(N-isopropylacrylamide)-based polymer particles were synthesised, and screened for the adsorption of human immunoglobulin G (hIgG). At pH 9 the adsorption on microgel particles was strongly affected by temperature, approximately 40 mg hIgG/g support (90% of initial hIgG) being adsorbed at 40 degrees C but only 10% of initial hIgG at 25 degrees C. At pH 5 the maximum adsorbed amount (20 mg hIgG/g support) was similar for both temperatures. The adsorption of hIgG on to charged poly(methyl methacrylate)/poly(N-isopropylacrylamide) core-shell latexes was negligible (5-10 mg hIgG/g support) at the same temperature and pH conditions. The lower adsorption of hIgG onto the core-shell particles is explained by steric interactions due to the small size of the shell.

An artificial protein L for the purification of immunoglobulins and fab fragments by affinity chromatography.

The development and characterization of an artificial protein L (PpL) for the affinity purification of antibodies is described. Ligand 8/7, which emerged as the lead from a de novo designed combinatorial library of ligands, inhibits the interaction of PpL with IgG and Fab by competitive ELISA and shows negligible binding to Fc. The ligand 8/7 adsorbent (Ka approximately 10(4) M(-1)) compared well with PpL in binding to immunoglobulins from different classes and sources and, in addition, bound to IgG1 with K and lambda isotypes (92% and 100% of loaded protein) and polyclonal IgG from sheep, cow, goat and chicken. These properties were also reflected in the efficient isolation of immunoglobulins from crude samples.

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%.

Antibodies and genetically engineered related molecules: production and purification.

Antibodies and antibody derivatives constitute 20 % of biopharmaceutical products currently in development, and despite early failures of murine products, chimeric and humanized monoclonal antibodies are now viable therapeutics. A number of genetically engineered antibody constructions have emerged, including molecular hybrids or chimeras that can deliver a powerful toxin to a target such as a tumor cell. However, the general use in clinical practice of antibody therapeutics is dependent not only on the availability of products with required efficacy but also on the costs of therapy. As a rule, a significant percentage (50-80%) of the total manufacturing cost of a therapeutic antibody is incurred during downstream processing. The critical challenges posed by the production of novel antibody therapeutics include improving process economics and efficiency, to reduce costs, and fulfilling increasingly demanding quality criteria for Food and Drug Administration (FDA) approval. It is anticipated that novel affinity-based separations will emerge from the development of synthetic ligands tailored to specific biotechnological needs. These synthetic affinity ligands include peptides obtained by synthesis and screening of peptide combinatorial libraries and artificial non-peptidic ligands generated by a de novo process design and synthesis. The exceptional stability, improved selectivity, and low cost of these ligands can lead to more efficient, less expensive, and safer procedures for antibody purification at manufacturing scales. This review aims to highlight the current trends in the design and construction of genetically engineered antibodies and related molecules, the recombinant systems used for their production, and the development of novel affinity-based strategies for antibody recovery and purification.

A new method for the screening of solid-phase combinatorial libraries for affinity chromatography.

A new methodology for the rapid assessment of affinity ligands synthesized by combinatorial solid-phase chemistry is reported. This screening strategy utilizes the target protein conjugated to FITC, and represents an almost universal technique for the preliminary screening of solid-phase combinatorial libraries. The assessment of a triazine-scaffolded solid-phase combinatorial library of ligands, designed to bind to human IgG, was performed with FITC-human IgG, and the results compared with those obtained by conventional affinity chromatographic screening assays. The effect of different molar conjugation ratios of FITC-IgG (F/P) was evaluated. Independently of the F/P ratio, no false negative results were observed, although lower F/P ratios diminished non-specific interactions and the number of false positives. The nature of the substituents on the triazine scaffold was not related to the number of false positive IgG-binding ligands. The reproducibility of the FITC technique, using FITC-human IgG conjugates with low F/P ratio (F/P=2), was also evaluated. The FITC-based technique proved to be efficient and accurate in the identification of strongly binding ligands (binding >50% of loaded protein, by standard affinity chromatographic assays), and is envisaged as a versatile and cost-effective method to screen other systems, and evaluate several binding/elution conditions at small-scale, prior to scale-up to standard affinity chromatography.

Evaluation of inducible promoters on the secretion of a ZZ-proinsulin fusion protein in Escherichia coli.

Four inducible promoters, uspA, uspB, lacUV5 and malK, were evaluated in the expression of the fusion protein ZZ-proinsulin by Escherichia coli. The aim was to select for their effects on the most appropriate expression system (promoter and culture medium) for secretion of ZZ-proinsulin to the periplasmic space and culture medium. All the expression vectors contained the RNase III cleavage site to ensure that the mRNA translation rate remained independent of 5'-untranslated regions thus making promoter strength comparisons more accurate. The highest ZZ-proinsulin secretion yields were 6.2 mg/g of dry cell weight in the periplasmic space and 2.6 mg/g of dry cell weight in the culture medium using the malK promoter. It was also demonstrated that the use of M9 minimal medium favours secretion.