Single-step purification of equine chorionic gonadotrophin directly from plasma using affinity chromatography.

Equine chorionic gonadotrophin (eCG) is a hormone widely used in superovulation protocols because of its follicle-stimulating action, which increases reproductive efficiency in animals of productive interest. It contains 45% carbohydrate, 10% of which is N-acetylneuraminic acid (sialic acid). The eCG purification procedures from equine serum or plasma are mainly based on chromatographic methods. However, before these procedures, it is necessary to follow sample pre-conditioning steps, such as several precipitation stages and/or ultrafiltration/diafiltration processes. In this work, an efficient affinity chromatographic matrix for eCG purification directly from plasma was developed. The matrix consisted of chitosan mini-spheres with immobilized wheat germ agglutinin (WGA). The matrix allowed 98% adsorption of eCG directly from plasma without any pre-treatment with an overall yield of around 60%. The matrix chosen was able to maintain the efficient performance of the purification process for three consecutive cycles. Also, the process was scaled-up 500 times in volume and tested over seven consecutive cycles maintaining its chromatographic performance. The results presented here suggest the potential application of this matrix to one-step purification of eCG from plasma.

Recombinant human follicle stimulating hormone purification by a short peptide affinity chromatography.

Peptide KVPLITVSKAK was selected to design a synthetic ligand for affinity chromatography purification of recombinant human follicle stimulating hormone (rhFSH), based on the interaction of the hormone with the exoloop 3 of its receptor. The peptide was acetylated to improve its stability to degradation by exopeptidases. A cysteine was incorporated at the C-termini to facilitate its immobilization to the chromatographic activated SulfoLink agarose resin. A sample of crude rhFSH was loaded to the affinity column, using 20 mM sodium phosphate, 0.5 mM methionine, and pH 5.6 and 7.2 as adsorption and elution buffers, respectively. The dynamic capacity of the matrix was 54.6 mg rhFSH/mL matrix and the purity 94%. The percentage of oxidized rhFSH was 3.4%, and that of the free subunits was 1.2%, both in the range established by the European Pharmacopeia, as also were the sialic acid content and the isoforms profile.

Sulfanilic acid-modified chitosan mini-spheres and their application for lysozyme purification from egg white.

A cation exchange matrix with zwitterionic and multimodal properties was synthesized by a simple reaction sequence coupling sulfanilic acid to a chitosan based support. The novel chromatographic matrix was physico-chemically characterized by ss-NMR and ζ potential, and its chromatographic performance was evaluated for lysozyme purification from diluted egg white. The maximum adsorption capacity, calculated according to Langmuir adsorption isotherm, was 50.07 ± 1.47 mg g-1 while the dissociation constant was 0.074 ± 0.012 mg mL-1 . The process for lysozyme purification from egg white was optimized, with 81.9% yield and a purity degree of 86.5%, according to RP-HPLC analysis. This work shows novel possible applications of chitosan based materials. The simple synthesis reactions combined with the simple mode of use of the chitosan matrix represents a novel method to purify proteins from raw starting materials. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:387-396, 2018.

Affinity chromatography matrices for depletion and purification of casein glycomacropeptide from bovine whey.

Casein glycomacropeptide (CMP) is a 64- amino acid peptide found in cheese whey, which is released after κ-casein specific cleavage by chymosin. CMP lacks aromatic amino acids, a characteristic that makes it usable as a nutritional supplement for people with phenylketonuria. CMP consists of two nonglycosylated isoforms (aCMP A and aCMP B) and its different glycosylated forms (gCMP A and gCMP B). The most predominant carbohydrate of gCMP is N-acetylneuraminic acid (sialic acid). Here, we developed a CMP purification process based on the affinity of sialic acid for wheat germ agglutinin (WGA). After formation of chitosan beads and adsorption of WGA, the agglutinin was covalently attached with glutaraldehyde. Two matrices with different WGA density were assayed for CMP adsorption. Maximum adsorption capacities were calculated according to the Langmuir model from adsorption isotherms developed at pH 7.0, being 137.0 mg/g for the matrix with the best performance. In CMP reduction from whey, maximum removal percentage was 79% (specifically 33.7% of gCMP A and B, 75.8% of aCMP A, and 93.9% of aCMP B). The CMP was recovered as an aggregate with an overall yield of 64%. Therefore, the matrices developed are promising for CMP purification from cheese whey. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:171-180, 2017.

Insect Larvae: A New Platform to Produce Commercial Recombinant Proteins.

In Biotechnology, the expression of recombinant proteins is a constantly growing field and different hosts are used for this purpose. Some valuable proteins cannot be produced using traditional systems. Insects from the order Lepidoptera infected with recombinant baculovirus have appeared as a good choice to express high levels of proteins, especially those with post-translational modifications. Lepidopteran insects, which are extensively distributed in the world, can be used as small protein factories, the new biofactories. Species like Bombyx mori (silkworm) have been analyzed in Asian countries to produce a great number of recombinant proteins for use in basic and applied science and industry. Many proteins expressed in this larva have been commercialized. Several recombinant proteins produced in silkworms have already been commercialized. On the other hand, species like Spodoptera frugiperda, Heliothis virescens, Rachiplusia nu, Helicoverpa zea and Trichoplusia ni are widely distributed in both the occidental world and Europe. The expression of recombinant proteins in larvae has the advantage of its low cost in comparison with insect cell cultures. A wide variety of recombinant proteins, including enzymes, hormones and vaccines, have been efficiently expressed with intact biological activity. The expression of pharmaceutically proteins, using insect larvae or cocoons, has become very attractive. This review describes the use of insect larvae as an alternative to produce commercial recombinant proteins.

Expression and functional characterization of a C-7 cholesterol desaturase from Tetrahymena thermophila in an insect cell line.

Tetrahymena thermophila transforms exogenous cholesterol into pro-vitamin D3 (7-dehydrocholesterol) with remarkable efficiency in a one-step reaction carried out by a C-7 cholesterol desaturase. The enzyme DES7 is encoded by the gene TTHERM_00310640, identified with RNAi and gene knock-out experiments, but has not yet been heterologously expressed actively in any organism. A model derived from its amino acid sequence classified DES7p as a Rieske-type oxygenase with transmembrane localization. The protein has catalytic activity, sequence and topological similarity to DAF-36/Neverland proteins involved in the synthesis of steroid hormones in insects and nematodes. Due to their structural and functional similarity, we analyzed the expression of a codon optimized DES7 gene from Tetrahymena in the insect Sf9 cell line, identified and measured the steroid metabolites formed, and extended the actual knowledge on its localization. We found that the accumulation of 7-dehydrocholesterol could be increased 16-40-fold in Spodopterafrugiperda, depending on physiological conditions, by overexpression of T. thermophila DES7. The protein was detected in the microsomal fraction, in accordance with previous reports. Although the electron transfer chain for Des7p/DAF-36/Neverland Rieske-type oxygenases is presently unknown, we identified possible donors in the ciliate and insect genomes by bioinformatic analysis. In spite of the large evolutionary distance between S. frugiperda and T. thermophila, the results indicate that there is significant functional conservation of the electron donors, since the ciliate's sterol desaturase can function in the context of the insect electron transport system. The results achieved demonstrate that DES7 is the first gene from a ciliate, coding for a microsomal enzyme, expressed in active form in an insect cell line.

A positive cooperativity binding model between Ly49 natural killer cell receptors and the viral immunoevasin m157: kinetic and thermodynamic studies.

Natural killer (NK) cells discriminate between healthy and virally infected or transformed cells using diverse surface receptors that are both activating and inhibitory. Among them, the homodimeric Ly49 NK receptors, which can adopt two distinct conformations (backfolded and extended), are of particular importance for detecting cells infected with mouse cytomegalovirus (CMV) via recognition of the viral immunoevasin m157. The interaction of m157 with activating (Ly49H) and inhibitory (Ly49I) receptors governs the spread of mouse CMV. We carried out kinetic and thermodynamic experiments to elucidate the Ly49/m157 binding mechanism. Combining surface plasmon resonance, fluorescence anisotropy, and circular dichroism (CD), we determined that the best model to describe both the Ly49H/m157 and Ly49I/m157 interactions is a conformational selection mechanism where only the extended conformation of Ly49 (Ly49*) is able to bind the first m157 ligand followed by binding of the Ly49*/m157 complex to the second m157. The interaction is characterized by strong positive cooperativity such that the second m157 binds the Ly49 homodimer with a 1000-fold higher sequential constant than the first m157 (∼10(8) versus ∼10(5) M(-1)). Using far-UV CD, we obtained evidence for a conformational change in Ly49 upon binding m157 that could explain the positive cooperativity. The rate-limiting step of the overall mechanism is a conformational transition in Ly49 from its backfolded to extended form. The global thermodynamic parameters from the initial state (backfolded Ly49 and m157) to the final state (Ly49*/(m157)2) are characterized by an unfavorable enthalpy that is compensated by a favorable entropy, making the interaction spontaneous.

Bovine lactoferrin purification from whey using Yellow HE-4R as the chromatographic affinity ligand.

The worldwide production of whey increases by around 186 million tons each year and it is generally considered as a waste, even when several whey proteins have important economic relevance. For its valorization, inexpensive ligands and integrated chromatography methods need to be developed for specific and low-cost protein purification. Here, we describe a novel affinity process with the dye Yellow HE-4R immobilized on Sepharose for bovine lactoferrin purification. This approach based on a low-cost ligand showed an efficient performance for the recovery and purification of bovine lactoferrin directly from whey, with a yield of 71% and a purification factor of 61.

Recombinant protein purification using complementary peptides as affinity tags.

Affinity tags have become highly popular tools for purifying recombinant proteins from crude extracts by affinity chromatography. Besides, short peptides are excellent ligands for affinity chromatography, as they are not likely to cause an immune response in case of leakage into the product, they are more stable than antibodies to elution and cleaning conditions and they usually have very acceptable selectivity. Hydropathically complementary peptides designed de novo show enough selectivity to be used successfully as peptide ligands for protein purification from crude extracts. Recognition specificity and selectivity in the interaction between the complementary peptide pair His-Leu-Leu-Phe-Pro-Ile-Ile-Ile-Ala-Ala-Ser-Leu and Lys-Asn-Tyr-Pro-Lys-Lys-Lys-Met-Glu-Lys-Arg-Phe have been demonstrated by other authors. In this work, we designed a recombinant protein purification method using a peptide affinity tag that binds to a peptide-binding partner immobilized on a chromatographic matrix. The enhanced green fluorescent protein expressed (EGFP) in Escherichia coli was used as the model. The peptide Gly-Gly-Gly-His-Leu-Leu-Phe-Pro-Ile-Ile-Ile-Ala-Ala-Ser-Leu was synthesized by solid phase using the Fmoc chemistry and immobilized in NHS-Sepharose (PC-Sepharose). Gly residues were added as a spacer arm at the N terminus. The EGFP was expressed either with the fusion tag Lys-Asn-Tyr-Pro-Lys-Lys-Lys-Met-Glu-Lys-Arg-Phe on the C terminus (EGFP-CPTag) or without any fusion tag. After cell disruption, the extract was directly applied to the PC-Sepharose column equilibrated with 20mM sodium phosphate buffer, pH 7.0. The adsorbed EGFP-CPTag was then eluted with 1M Tris. The yield was 98% and the purification factor 4.6. By contrast, EGFP without tag pass through without interacting with the PC-Sepharose column. The method designed can be applied for the purification of other recombinant proteins.

Efficient wheat germ agglutinin purification with a chitosan-based affinity chromatographic matrix.

An efficient affinity chromatographic matrix based on chitosan for wheat germ agglutinin (WGA) purification was developed. The matrices assayed consisted of chitosan mini-spheres cross-linked with epichlorhydrin 45, 250 or 500 mM. The maximum adsorption capacity of pure WGA - calculated from the corresponding isotherms - was between 43.2 and 48.9 mg/g at pH 5.0 and between 16.6 and 27.6 mg/g at pH 8.5. However, the adsorption of agglutinin from wheat germ extract was higher at pH 8.5. In addition, 0.5 g of mini-spheres cross-linked with epichlorhydrin 250 mM adsorbed 94.5% of the WGA present in 5 mL of the concentrated extract. Acetic acid was able to elute 100% of the adsorbed WGA. The purity of the WGA obtained was greater than 95% and the purification factor was 56.8. The matrix was able to maintain an efficient performance of the purification process for three consecutive cycles. A new method to monitor the purification process by RP-HPLC was developed.

Predominantly Cytoplasmic Localization in Yeast of ASR1, a Non-Receptor Transcription Factor from Plants.

The Asr gene family (named after abscisic acid, stress and ripening), currently classified as a novel group of the LEA superfamily, is exclusively present in the genomes of seed plants, except for the Brassicaceae family. It is associated with water-deficit stress and is involved in adaptation to dry climates. Motivated by separate reports depicting ASR proteins as either transcription factors or chaperones, we decided to determine the intracellular localization of ASR proteins. For that purpose, we employed an in vivo eukaryotic expression system, the heterologous model Saccharomyces cerevisiae, including wild type strains as well as mutants in which the variant ASR1 previously proved to be functionally protective against osmotic stress. Our methodology involved immunofluorescence-based confocal microscopy, without artificially altering the native structure of the protein under study. Results show that, in both normal and osmotic stress conditions, recombinant ASR1 turned out to localize mainly to the cytoplasm, irrespective of the genotype used, revealing a scattered distribution in the form of dots or granules. The results are discussed in terms of a plausible dual (cytoplasmic and nuclear) role of ASR proteins.