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.     

Mineralocorticoid radioreceptor assay: application to adrenal-regeneration hypertension.

Identification of unknown hormones has traditionally involved utilizing a bioassay to initially detect the hormone and to follow its purification. However, radioreceptor assays may be more useful for this purpose by offering greater sensitivity and precision. A mineralocorticoid radioreceptor assay has been developed for use in conjunction with chromatographic separation of a urinary extract to detect the presence of unknown urinary mineralocorticoids. This assay utilizes competition of the unknown steroid and aldosterone for rat renal cytoplasmic mineralocorticoid receptors to enable mineralocorticoid quantitation in aldosterone equivalents. This assay provides 100 fold increase in sensitivity and a significant increase in precision over the commonly used adrenalectomized rat bioassay. The mineralocorticoid radioreceptor assay has been utilized to assay mineralocorticoid activity in chromatographic fractions of a urinary extract from rats with regenerating adrenals. A large area of mineralocorticoid radioreceptor activity has been identified which possibly represents an unknown mineralocorticoid contributing to the etiology of adrenal regeneration hypertension. This assay is applicable to other syndromes of postulated unknown mineralocorticoid excess, such as human low renin essential hypertension. In addition, similar radioreceptor assays are applicable for the initial detection of any type of hormone activity and for the subsequent purification and identification of this hormone.     

Expression of human insulin-like growth factor I in bacteria: use of optimized gene fusion vectors to facilitate protein purification

Several fusions between the gene for human insulin-like growth factor I (IGF-I) and the genes for different IgG-binding fragments of staphylococcal protein A were assembled and compared regarding expression, secretion, and purification of the peptide hormone. After IgG affinity purification of the fusion proteins from the growth medium of Staphylococcus aureus or Escherichia coli, native IGF-I was released by cleavage of an Asn-Gly peptide bond with hydroxylamine. An optimized expression system based on a modified synthetic IgG-binding domain (z), resistant to hydroxylamine, gave the highest yield of fusion protein. After cleavage, the hormone could be separated from the IgG-binding moiety and from noncleaved fusion protein by a second passage through the IgG affinity column. The biological activity and the purity of the IGF-I obtained were confirmed by a radioreceptor assay, N-terminal sequence analysis, polyacrylamide gel electrophoresis, isoelectric focusing, and high-performance liquid chromatography.     

Soluble expression and purification of synthetic human bone morphogenetic protein-2 in Escherichia coli

A 345-bp gene that encodes human bone morphogenetic protein-2 (hBMP-2) has been synthesized. The codon usage of the resulting gene was modified to include those triplets that are utilized in highly expressed Escherichia coli genes. The hBMP-2 gene was efficiently expressed in E. coli as a soluble and active protein. Since the recombinant hBMP-2 was readily solublized, no further solublization steps were required throughout purification. No additional tagging residues were introduced into the synthetic hBMP-2 gene product. The developed synthetic gene is a promising approach for scaling-up the soluble expression of hBMP-2.     

Conservation of the DNA binding domain and other properties between porcine and rat glucocorticoid receptors

Activated glucocorticoid receptor protein (GCR) was partially purified from porcine liver cytosol by sequential chromatography on phosphocellulose and DNA-cellulose using a modification of a protocol developed for purification of rat GCR. This partially purified preparation, when separated by SDS-polyacrylamide gel electrophoresis and immunoblotted, indicated that a Mr = 94,000 protein band cross-reacts with a monoclonal antibody against rat GCR. A nitrocellulose filter binding assay showed that both the partially purified porcine and rat GCRs interact specifically with a cloned synthetic 24 base pair deoxyoligonucleotide containing the GCR binding sequence in the first intron of the human growth hormone (hGH) gene. This specific protein-DNA interaction is blocked by a single base pair change in the binding site. All three putative domains of the GCR molecule: the steroid binding, immunoreactive, and DNA binding have been conserved between two divergent species.     

Site-specific antibodies to human erythropoietin: immunoaffinity purification of urinary and recombinant hormone

The 19-amino acid domain Ala111----Pro129 of human erythropoietin was identified as an accessible surface antigen based on the binding of radio-iodinated and of unmodified hormone to antibodies prepared against a synthetic peptide of homologous sequence. The specificity and affinity of this binding was sufficient to provide for the use of anti-peptide antibodies in the preparation of an immunosorbent for the purification of urinary, and of recombinant human erythropoietin. Immobilization of anti-peptide antibodies using agarose activated either with CNBr or with N-hydroxysuccinimido groups largely inactivated binding sites for erythropoietin. In contrast, antibodies crosslinked to N-acetyl-DL-homocysteine agarose through the hetero-bifunctional reagent succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate retained their antigen-binding capacity virtually completely and provided a superior immunosorbent for hormone. Urinary erythropoietin with a specific bioactivity of 100 U/A280 was prepared initially by chromatography on phenyl-Sepharose. Subsequent immunoaffinity chromatography resulted in a 350-fold purification with 46.2% recovery yielding erythropoietin with a specific bioactivity of 35,200 U/A280 (44,300 U/mg). Radioiodination of this purified protein and subsequent SDS-polyacrylamide gel electrophoresis indicated that this preparation contained a single major component (Mr 30,000) which co-migrated in gels with unmodified biologically active hormone. Recombinant erythropoietin, which was prepared by the cloning of the human erythropoietin gene and its expression in COS cells using the SV40-derived vector pSV2, was purified by the same scheme. Chromatography on phenyl-Sepharose of medium derived from transfected cells (400 U/ml, 170 U/A280) provided for a 3.6-fold purification of recombinant hormone with an apparent recovery of 122%. This erythropoietin bound to the anti-peptide antibody gel and was purified to a specific bioactivity of 10,370 U/A280 with 55% recovery. The procedure described here for attaching antibodies to a solid support maximizes their antigen-binding capacity and is generally applicable. The development of an anti-peptide immunosorbant for human erythropoietin provides a valuable means for isolating hormone for use in studies of its receptor and its presently unresolved mechanism of action.     

Analysis of Hormone–Receptor Interaction Sites Using Synthetic Peptides: Receptor Binding Regions of the α-Subunit of Human Choriogonadotropin

Synthetic peptides are valuable tools for determining sites of interaction between hormones and their receptors. We have learned much about linear receptor binding regions of the glycoprotein hormone human choriogonadotropin (hCG) using synthetic peptides corresponding to its primary sequence. Of paramount importance in any study using synthetic peptides as a tool to investigate protein structure are an efficient means of peptide purification and a definitive measure of peptide purity and composition. Purification is easily achieved for all but the most hydrophobic peptides using preparative reverse-phase HPLC. Of the methods available for analysis of peptide purity, mass spectrometry is perhaps the most useful and often most rapid approach. Other essential components of studies involving synthetic peptides and hormone binding are reproducible hormone labeling, receptor preparations, and bioassays. The ability of peptides to compete with hCG for binding to specific receptors is tested in radioreceptor binding assays and bioassays with transformed Leydig cell lines. These cells express the lutropin receptor that is coupled to a measurable endpoint such as cAMP or steroid production. The conditions for these assays are optimized for rapid and accurate measurement of peptide activity. Since the three-dimensional structure of hCG is not known, a systematic approach to the identification of potential receptor binding sites is advocated. First, a comprehensive analysis using synthetic overlapping peptides of uniform length that span the entire sequence of the α-subunit is employed. This approach is an effective means for surveying the entire subunit for receptor binding sites. Next, the boundaries of the active regions are delimited by a series of nested peptides sequentially shortened in length. The importance of each residue within the delimited binding regions is then studied using a series of peptides containing single alanine substitutions. Finally, modifications to enhance activity of the synthetic peptides are further made on the basis of data from alanine substitution studies, circular dichroic analysis, and molecular modeling. These studies provide valuable information to aid in the design of synthetic hormone analogs and for further analysis of the structure–function of hCG via site-directed mutagenesis.     

Identification of a surface epitope of human erythropoietin with anti-peptide antibodies

Antibodies reactive with human erythropoietin were isolated from the serum of rabbits immunized with a twenty-six amino acid synthetic polypeptide corresponding to a proposed NH2-terminal sequence of the hormone. As shown by inhibition with peptide fragments, those antibodies that bound to erythropoietin recognized the (8-15) domain, strongly suggesting tht this region is exposed on the hormone's surface. This was confirmed by affinity purification of these antibodies on immobilized fragment (8-15). These results provide insight into the tertiary structure of human erythropoietin and suggest uses for the sequence-specific antibodies in labeling the hormone.     

Utilization of protein splicing for purification of the human growth hormone

Developing simple and reliable methods to purify recombinant proteins is among the most important problems of modern biotechnology. It is of particular interest to take advantage of protein splicing for this purpose. Affinity tagging of inteins allows the use of regular protocols for protein purification. Autocatalytic excision of the tagged intein yields the pure protein lacking N-terminal formylmethionine. A new purification technique was developed on the basis of protein splicing for the human growth hormone. The Mxe GyrA intein with the histidine tag or cellulose-binding domain was used as a self-removable affinity unit. The resulting two-step purification protocol makes it possible to obtain the human growth hormone having the native N terminus with minimal losses.     

The potential of adipokinetic hormone to teach us about neuropeptides

Genetic manipulation and purification techniques are facilitating research into the biology of arthropod neuropeptides. The red pigment concentrating hormone (RPCH)/ adipokinetic hormone (AKH) family are a conserved group of peptides which were first recognized for their hormonal activities. Biosynthesis of AKH in the grasshopper seems to proceed via a large protein precursor (12 kDa) in the cell body of the neuron, which implies precise coordination of synthesis and neural activity. Beginning with a 10 amino acid stretch of known precursor protein sequence, an oligonucleotide primer approach is being used to clone a recombinant AKH gene. Once achieved, this will allow fundamental questions on the biochemistry and physiology of neuropeptides to be addressed. Such work in an arthropod system raises the intriguing possibility of studying neuropeptide level and synthetic activity in single, defined neurons.     

Expression and characterization of a recombinant human parathyroid hormone secreted by Escherichia coli employing the staphylococcal protein A promoter and signal sequence

Human parathyroid hormone (hPTH) is a peptide hormone consisting of 84 amino acids (hPTH(1-84)). Employing the promoter and signal sequence of Staphylococcus aureus-protein A we have expressed hPTH in Escherichia coli. The expressed proteins are excreted to the growth medium, allowing for rapid and easy purification of the desired products. By amino acid sequence analysis and mass spectrometry, we have shown that the major excreted product is correctly processed human identical hPTH(1-84). The purified recombinant hPTH(1-84) stimulates adenylate cyclase activity in rat osteosarcoma cell membranes to exactly the same extent as synthetic parathyroid hormone standards, indicating that the recombinant product has full biological activity.     

Analysis of protein digests by capillary high-performance liquid chromatography and on-line fast atom bombardment mass spectrometry

An HPLC system incorporating a packed capillary C18 column has been utilized for high sensitivity peptide mapping and preparative collection for protein sequencing. This system combined with a Frit-FAB mass spectrometer interface also provides the ability to obtain molecular ions for peptides of enzymatically digested proteins in the time it takes to obtain an HPLC chromatogram. The low flow rates permit introduction of the entire column effluent into the mass spectrometer. Detection limits of 0.5-5 pmol are routine. Proteolytic digests of recombinant human methionyl growth hormone and protein carboxyl methyltransferase have been used to demonstrate the HPLC and mass spectrometer performance.     

Purification of antibodies using the synthetic affinity ligand absorbent MAbsorbent A2P

A protocol for the purification of polyclonal antibodies from ovine serum using the synthetic protein A absorbent MAbsorbent A2P is described. Clarified serum is loaded directly onto the affinity column without prior adjustment and albumin and unwanted serum components are washed from the column using a sodium octanoate buffer before elution of bound antibodies. MAbsorbent A2P was shown to bind approximately 27 mg ml(-1) of polyclonal immunoglobulin under overloading conditions, with eluted IgG purities of >90% and minor levels of albumin (approximately 1%). The anticipated time required to complete the purification protocol is 6-7 h. Although the protocol is similar to methods utilized for antibody purification using chromatography with protein A derived from the cell wall of the microorganism Staphylococcus aureus or protein G from Streptococcus as the affinity ligands, affinity absorbents based on synthetic ligands offer a number of advantages to compounds derived from biological sources, in particular robustness, relatively low cost, ease of sanitization and, in principle, lack of biological contamination.     

A modified metal-ion affinity chromatography procedure for the purification of histidine-tagged recombinant proteins expressed in Drosophila S2 cells

We have developed a modified method of immobilized metal-ion affinity chromatography (IMAC) that can be used for the purification of histidine-tagged proteins from conditioned medium containing free copper ions. Classical methods of IMAC purification, using resins such as Ni-NTA, have proven inefficient for this type of purification and require multiple steps due to the interference of divalent copper ions with the binding of His-tagged protein to the charged resin. In contrast, this modified IMAC procedure, using chelating Sepharose instead of Ni-NTA, enables efficient purification from copper-containing medium in a single step. This method appears to rely upon a preferential interaction of protein-copper complexes with immobilized chelating resin. We have utilized this method to purify active, His-tagged murine interleukin 12 from the conditioned medium of Drosophila S2 cells coexpressing recombinant p40 and His-tagged p35 subunits and for the purification of the extracellular domain of the erythropoietin receptor. This method should be applicable to the purification of a wide variety of His-tagged fusion proteins expressed in Drosophila cells and in other systems where free metal ions are present.     

Isolation and detection of human IgA using a streptococcal IgA-binding peptide

Bacterial proteins that bind to the Fc part of IgG have found widespread use in immunology. A similar protein suitable for the isolation and detection of human IgA has not been described. Here, we show that a 50-residue synthetic peptide, designated streptococcal IgA-binding peptide (Sap) and derived from a streptococcal M protein, can be used for single-step affinity purification of human IgA. High affinity binding of IgA required the presence in Sap of a C-terminal cysteine residue, not present in the intact M protein. Passage of human serum through a Sap column caused depletion of >99% of the IgA, and elution of the column allowed quantitative recovery of highly purified IgA, for which the proportions of the IgA1 and IgA2 subclasses were the same as in whole serum. Moreover, immobilized Sap could be used for single-step purification of secretory IgA of both subclasses from human saliva, with a recovery of approximately 45%. The Sap peptide could also be used to specifically detect IgA bound to Ag. Together, these data indicate that Sap is a versatile Fc-binding reagent that may open new possibilities for the characterization of human IgA.     

Generation of recombinant cytoplasmic domain of epidermal growth factor receptor with intrinsic protein tyrosine kinase activity

We have generated a recombinant baculovirus using the high expression vector pVL941 containing the complementary DNA encoding the intracellular domain of the human epidermal growth factor receptor (EGFR-IC). Upon infection of Spodoptera frugiperda insect cells, protein tyrosine kinase-active EGFR-IC was produced. The expressed protein has a molecular weight of 61,000 and is specifically recognized by antibodies directed against peptides representing different regions of human EGFR-IC. Upon sonication of infected cells, EGFR-IC was detected in both the soluble and insoluble fractions of the cell lysate. About 20-50% of the expressed EGFR-IC was soluble. Metabolic labeling and protein analyses showed that EGFR-IC comprised 7% of newly synthesized proteins in the cytoplasmic lysate and 0.1-0.2% of the total soluble protein. We have used a three-step purification procedure (fast-Q-Sepharose and phenyl-Sepharose column chromatographies and 30% ammonium sulfate precipitation) to purify EGFR-IC to 85% purity with 15-20% recovery from the initial soluble lysate. A yield of 3-4 mg of purified EGFR-IC has been consistently produced from 20 roller bottles with 2-4 x 10(8) infected cells/bottle. The tyrosine kinase activity was retained through purification. The enzyme demonstrated much higher autophosphorylation activity in the presence of Mn2+ than Mg2+. Phosphopeptide mapping revealed the same autophosphorylation sites utilized by EGFR-IC as those identified in wild-type EGFR. EGFR-IC-catalyzed phosphorylation of either a synthetic peptide representing the major autophosphorylation site or angiotensin II showed that the baculovirus-expressed EGFR-IC exhibits similar enzymatic kinetic characteristics to the intact activated EGFR kinase.     

蛋白质层析用离子交换和疏水作用层析介质的发展概况

层析是蛋白质纯化的关键技术之一 ,作为层析技术的核心———层析介质一直以来是层析技术研究的一个热点。近年来 ,越来越多的新型层析介质被开发出来 ,如粒度均匀的交联多糖、人工合成的大孔聚合物、触角型吸附剂、软胶包裹在硬胶表面等介质。主要介绍应用较为广泛的IEC和HIC介质的组成、特性及其在蛋白质纯化中的应用 ,还研究了与HIC技术相关的两种新技术 :亲硫层析和疏水电荷诱导层析 (HCIC) ,重点介绍了HCIC的介质及其应用 ,同时也讨论了在蛋白质纯化中应用的三相纯化策略 (富集、中间纯化和精制 )。结合我国的实际情况 ,就当前蛋白质纯化的离子交换和疏水层析介质面临的挑战和未来的发展进行讨论并提出了建议     

Purification of growth hormones by reversed-phase high-performance liquid chromatography

Reversed-phase high-performance liquid chromatography (HPLC) on a column of Radial-Pak C₁₈ cartridge was utilized for the purification of a variety of growth hormone (GH) proteins from mammalian, avian, amphibian and fish pituitary glands. Recovery of GH from pituitary glands of up to 0.43% of total protein was obtained with a high degree of homogeneity as revealed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The HPLC-purified GHs show reactions of identity or near identity by immuno-diffusion studies on agar gel. This method offers a convenient and rapid purification of vertebrate GH on an analytical or preparative scale.     

Isolation of human proteasomes and putative proteasome-interacting proteins using a novel affinity chromatography method

The proteasome is the primary subcellular organelle responsible for protein degradation. It is a dynamic assemblage of 34 core subunits and many differentially expressed, transiently interacting, modulatory proteins. This paper describes a novel affinity chromatography method for the purification of functional human holoproteasome complexes using mild conditions. Human proteasomes purified by this simple procedure maintained the ability to proteolytically process synthetic peptide substrates and degrade ubiquitinated parkin. Furthermore, the entire purification fraction was analyzed by mass spectrometry in order to identify proteasomal proteins and putative proteasome-interacting proteins. The mild purification conditions maintained transient physical interactions between holoproteasomes and a number of known modulatory proteins. In addition, several classes of putative interacting proteins co-purified with the proteasomes, including proteins with a role in the ubiquitin proteasome system for protein degradation or DNA repair. These results demonstrate the efficacy of using this affinity purification strategy for isolating functional human proteasomes and identifying proteins that may physically interact with human proteasomes.