Targeting of bone morphogenetic protein complexes to heparin/heparan sulfate glycosaminoglycans in bioactive conformation.

Bone morphogenetic proteins (BMP) are powerful regulators of cellular processes such as proliferation, differentiation, and apoptosis. However, the specific molecular requirements controlling the bioavailability of BMPs in the extracellular matrix (ECM) are not yet fully understood. Our previous work showed that BMPs are targeted to the ECM as growth factor-prodomain (GF-PD) complexes (CPLXs) via specific interactions of their PDs. We showed that BMP-7 PD binding to the extracellular microfibril component fibrillin-1 renders the CPLXs from an open, bioactive V-shape into a closed, latent ring shape. Here, we show that specific PD interactions with heparin/heparan sulfate glycosaminoglycans (GAGs) allow to target and spatially concentrate BMP-7 and BMP-9 CPLXs in bioactive V-shape conformation. However, targeting to GAGs may be BMP specific, since BMP-10 GF and CPLX do not interact with heparin. Bioactivity assays on solid phase in combination with interaction studies showed that the BMP-7 PD protects the BMP-7 GF from inactivation by heparin. By using transmission electron microscopy, molecular docking, and site-directed mutagenesis, we determined the BMP-7 PD-binding site for heparin. Further, fine-mapping of the fibrillin-1-binding site within the BMP-7 PD and molecular modeling showed that both binding sites are mutually exclusive in the open V- versus closed ring-shape conformation. Together, our data suggest that targeting exquisite BMP PD-binding sites by extracellular protein and GAG scaffolds integrates BMP GF bioavailability in a contextual manner in development, postnatal life, and connective tissue disease.

Determination of Lactoferrin in Camel Milk by Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry Using an Isotope-Labeled Winged Peptide as Internal Standard.

An ultrahigh-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the determination of lactoferrin in camel milk based on the signature peptide. The camel lactoferrin was purified by heparin affinity chromatography and then used to screen tryptic signature peptides. The signature peptide was selected on the basis of sequence database search and identified from the tryptic hydrolysates of purified camel lactoferrin by ultrahigh-performance liquid chromatography and quadrupole time-of-flight tandem mass spectrometry. The pretreatment procedures included the addition of isotope-labeled winged peptide and the disposal of lipids and caseins followed by an enzymatic digestion with trypsin. Analytes were separated on an Acquity UPLC BEH 300 C18 column and then detected on a triple-quadrupole mass spectrometer in 7 min. The limits of detection and quantification were 3.8 mg kg-1 and 11 mg kg-1, respectively. The recoveries ranged from 74.5% to 103.6%, with relative standard deviations below 7.7%. The validated method was applied to determine the lactoferrin in ten samples collected from Xinjiang Province.

Heparin: role in protein purification and substitution with animal-component free material.

Heparin is a highly sulfated polysaccharide which belongs to the family of glycosaminoglycans. It is involved in various important biological activities. The major biological purpose is the inhibition of the coagulation cascade to maintain the blood flow in the vasculature. These properties are employed in several therapeutic drugs. Heparin's activities are associated with its interaction to various proteins. To date, the structural heparin-protein interactions are not completely understood. This review gives a general overview of specific patterns and functional groups which are involved in the heparin-protein binding. An understanding of the heparin-protein interactions at the molecular level is not only advantageous in the therapeutic application but also in biotechnological application of heparin for downstreaming. This review focuses on the heparin affinity chromatography. Diverse recombinant proteins can be successfully purified by this method. While effective, it is disadvantageous that heparin is an animal-derived material. Animal-based components carry the risk of contamination. Therefore, they are liable to strict quality controls and the validation of effective good manufacturing practice (GMP) implementation. Hence, adequate alternatives to animal-derived components are needed. This review examines strategies to avoid these disadvantages. Thereby, alternatives for the provision of heparin such as chemical synthesized heparin, chemoenzymatic heparin, and bioengineered heparin are discussed. Moreover, the usage of other chromatographic systems mimetic the heparin effect is reviewed.

N-Glycoproteomics of Human Seminal Plasma Glycoproteins.

Seminal plasma aids sperm by inhibiting premature capacitation, helping in the intracervical transport and formation of an oviductal sperm reservoir, all of which appear to be important in the fertilization process. Epitopes such as Lewis x and y are known to be present on seminal plasma glycoproteins, which can modulate the maternal immune response. It is suggested by multiple studies that seminal plasma glycoproteins play, largely undiscovered, important roles in the process of fertilization. We have devised a strategy to analyze glycopeptides from a complex, unknown mixture of protease-digested proteins. This analysis provides identification of the glycoproteins, glycosylation sites, glycan compositions, and proposed structures from the original sample. This strategy has been applied to human seminal plasma total glycoproteins. We have elucidated glycan compositions and proposed structures for 243 glycopeptides belonging to 73 N-glycosylation sites on 50 glycoproteins. The majority of the proposed glycan structures were complex type (83%) followed by high-mannose (10%) and then hybrid (7%). Most of the glycoproteins were either sialylated, fucosylated, or both. Many Lewis x/a and y/b epitopes bearing glycans were found, suggesting immune-modulating epitopes on multiple seminal plasma glycoproteins. The study also shows that large scale N-glycosylation mapping is achievable with current techniques and the depth of the analysis is roughly proportional to the prefractionation and complexity of the sample.

Overexpression and Purification of Mitogenic and Metabolic Fibroblast Growth Factors.

Fibroblast growth factors (FGFs) are proteins with a vast array of biological activity, such as cell development and repair, glucose and bile acid metabolisms, and wound healing. Due to their critical and diverse physiological functions, FGFs are believed to possess potential as therapeutic agents for many diseases and conditions that warrant further investigations. Thus, a simple, cost-efficient method to purify these biologically active signaling proteins is desirable. Herein, we introduce such techniques to purify FGFs that possess either high heparin-binding affinity or low to no heparin-binding affinity. This method takes advantage of the high affinity toward heparin sulfate from paracrine FGF1 to isolate the targeted protein. It also accounts for FGF members that have low heparin affinity, such as the metabolic FGFs, by introducing poly-histidine tags in the recombinant protein in combination with the immobilized metal affinity chromatography. Subsequently, the purified FGF products are separated from the other small protein by high-speed centrifugation. Products are then subjected to other biophysical experiments like SDS-PAGE, mass spectrometry, circular dichroism, intrinsic fluorescence, isothermal titration calorimetry, differential scanning calorimetry, and biological cell activity assay to confirm that the target proteins are purified with intact native conformation and no significant change in the intrinsic characteristics and biological activities.

Rapid and Robust Continuous Purification of High-Titer Hepatitis B Virus for In Vitro and In Vivo Applications.

Available treatments for hepatitis B can control the virus but are rarely curative. This led to a global initiative to design new curative therapies for the 257 million patients affected. Discovery and development of these new therapies is contingent upon functional in vitro and in vivo hepatitis B virus (HBV) infection models. However, low titer and impurity of conventional HBV stocks reduce significance of in vitro infections and moreover limit challenge doses in current in vivo models. Therefore, there is a critical need for a robust, simple and reproducible protocol to generate high-purity and high-titer infectious HBV stocks. Here, we outline a three-step protocol for continuous production of high-quality HBV stocks from supernatants of HBV-replicating cell lines. This purification process takes less than 6 h, yields to high-titer stocks (up to 1 × 1011 enveloped, DNA-containing HBV particles/mL each week), and is with minimal equipment easily adaptable to most laboratory settings.

Purification of the human fibroblast growth factor 2 using novel animal-component free materials.

This paper analyzes the use of animal-component free chromatographic materials for the efficient purification of the human fibroblast growth factor 2 (hFGF-2). hFGF-2 is produced in Escherichia coli and purified via three different chromatography steps, which include a strong cation exchange chromatography as a capture step, followed by heparin affinity chromatography and an anion exchange chromatography as a polishing step. The affinity chromatography step is based on the animal-derived material heparin. Chemically produced ligands provide a viable alternative to animal-derived components in production processes, since they are characterized by a defined structure which leads to reproducible results and a broad range of applications. The alternative ligands can be assigned to adsorber of the mixed-mode chromatography (MMC) and pseudo-affinity chromatography. Eight different animal-component free materials used as adsorbers in MMC or pseudo-affinity chromatography were tested as a substitute for heparin. The MMCs were cation exchangers characterized with further functional residues. The ligands of the pseudo-affinity chromatography were heparin-like ligands which are based on heparin's molecular structure. The alternative methods were tested as a capture step and in combination with another chromatographic step in the purification procedure of hFGF-2. In each downstream step purity, recovery and yield were analysed and compared to the conventional downstream process. Two types of MMC - the column ForesightTM NuviaTM cPrimeTM from Bio-Rad Laboratories and the column HiTrapTM CaptoTM MMC from GE Healthcare Life Sciences - can be regarded as effective animal-component free alternatives to the heparin - based adsorber.

Methods for Expression and Purification of Biologically Active Recombinant Progranulin.

Purifying progranulin may be useful in a variety of situations, for example, after it has been mutated or otherwise modified or when working with a species for which commercially produced progranulin is unavailable. A method to express and purify human progranulin is presented. Progranulin is transiently expressed in mammalian cells and isolated from their conditioned medium before purification by reversed-phase high-performance liquid chromatography (RP-HPLC). Human progranulin is used as an example, but the protocol can be applied to any other progranulin protein. Modifications of the expression-purification strategy for metabolic labeling of progranulin and analytical systems based on heparin-affinity chromatography are presented.

Purification of foot-and-mouth disease virus by heparin as ligand for certain strains.

The goal of this project was to develop an easily operable and scalable process for the recovery and purification of foot-and-mouth disease virus (FMDV) from cell culture. Heparin resins HipTrap Heparin HP and AF-Heparin HC-650 were utilized to purify FMDV O/HN/CHA/93. Results showed that the purity of AF-Heparin HC-650 was ideal. Then, the O/HN/CHA/93, O/Tibet/CHA/99, Asia I/HN/06, and A/CHA/HB/2009 strains were purified by AF-Heparin HC-650. Their affinity/virus recoveries were approximately 51.2%/45.8%, 71.5%/70.9%, 96.4%/73.5, and 59.5%/42.1%, respectively. During a stepwise elution strategy, the viral particles were mainly eluted at 300mM ionic strength peaks. The heparin affinity chromatography process removed more than 94% of cellular and medium proteins. Anion exchange resin Capto Q captured four FMD virus particles; 40% of binding proteins and 80%-90% of viral particles were eluted at 450mM NaCl. Moreover, ionic strength varied from 30 to 450mM had no effect on the immunity to FMDV. The results revealed that heparin sulfate may be the main receptor for CHA/99 strain attachment-susceptible cells. Heparin affinity chromatography can reach perfect results, especially when used as a ligand of the virus. Anion exchange is useful only as previous step for further purification.

Production of Recombinant CCN2 Protein by Mammalian Cells.

Recombinant CCN2 protein (rCCN2) is available from many companies; however, most of them are produced in E. coli. To investigate true functions of rCCN2, glycosylated protein with proper folding needs to be used. Therefore, we use rCCN2 produced by mammalian cells. Conditioned medium (CM) of HeLa cells stably transfected with a CCN2 expression vector are collected, and the recombinant CCN2 protein produced and secreted into the CM is purified by two-step chromatography, first with a heparin affinity column and then with an anti-CCN2 affinity column prepared with a monoclonal antibody against CCN2. The purified rCCN2 shows the bands of 36-38 kDa with sliver staining after gel electrophoresis, which can be confirmed by Western blotting. This chapter describes these methods in detail.