Co-expression and purification of recombinant human insulin-like growth factor II and insulin-like growth factor binding protein-6 in Pichia pastoris yeast

For the preparation of the complex of IGF-II and IGFBP-6, a co-expression vector containing two copies of human IGF-II and IGFBP-6 expression cassette was constructed with alcohol oxidase (AOX1) promoter and secretion signal sequence of alpha-factor, and transformed to Pichia pastoris yeast. Through a purification procedure involving anion-exchange chromatography and gel filtration, a complex of IGF-II with IGFBP-6 was obtained. An additional C-terminal sequence of IGFBP-6 (CS-BP6) was found to be bound to this complex. Dynamic light scattering showed that this complex was very stable and homogenous in solution. Western blotting based on non-reducing Tricine-SDS-PAGE indicated that IGF-II expression coupled with IGFBP-6 might significantly avoid the mispairing of disulfide bonds compared with the IGF-II expressed alone.     

Characterization of insulin-like growth factor binding protein-1 kinases from human hepatoma cells

The phosphorylation of insulin-like growth factor binding protein-I (IGFBP-1) alters its binding affinity for insulin-like growth factor I (IGF-I) and thus regulates the bioavailability of IGF-I for binding to the IGF-I receptor. The kinase(s) responsible for the phosphorylation of IGFBP-1 has not been identified. This study was designed to characterize the IGFBP-1 kinase activity in HepG2 human hepatoma cells, a cell line that secretes IGFBP-1 primarily as phosphorylated isoforms. IGFBP-1 kinase activity was partially purified from detergent extracts of the cells by phosphocellulose chromatography and gel filtration. Two kinases of approximate M_r 150,000 (peak I kinase) and M_r 50,000 (peak II kinase) were identified. Each kinase phosphorylated IGFBP-1 at serine residues that were phosphorylated by intact HepG2 cells. The kinases were distinct based on their differential sensitivity to inhibition by heparin (IC₅₀ = 2.5 and 16.5 μg/ml, peak I and II kinase, respectively) and inhibition by the isoquinoline sulfonamide CKI-7 (IC₅₀ = 50 μM and 100 μM, peak I and II kinase, respectively). In addition, a tenfold molar excess of nonradioactive GTP relative to [gamma-³²P]ATP lowered the incorporation of ³²P into IGFBP-1 by 80% when the reaction was catalyzed by the peak I kinase, whereas GTP had no effect on the reaction catalyzed by the peak II kinase. In the presence of polylysine, IGFBP-1 was radiolabeled by the partially purified kinase activity when [gamma-³²P]GTP served as the phosphate donor indicating the presence of casein kinase II activity. Furthermore, IGFBP-1 was phosphorylated by purified casein kinase I and casein kinase II at sites phosphorylated by the peak I and peak II kinases. Our data suggest that at least two kinases could be responsible for the phosphorylation of IGFBP-1 in intact HepG2 cells and that the kinases are related to the casein kinase family of protein kinases. © 1996 Wiley-Liss, Inc.     

Human insulin-like growth factor binding protein-6 is O-glycosylated.

Insulin-like growth factor binding protein-6 is abundant in cerebrospinal fluid and has a marked preferential binding affinity for IGF-II over IGF-I. The present study demonstrates that IGFBP-6 is O-glycosylated but not N-glycosylated. Carbohydrate analysis revealed the presence of approximately 20-30 carbohydrate residues/molecule. Galactosamine, galactose and sialic acid were most abundant, with glucosamine and fucose present in lower concentrations. Mannose was not detected. Enzymatic deglycosylation did not alter the high affinity of IGF binding protein-6 for IGF-II (Ka 4.4 +/- 2.2 x 10(11) M-1) or its preference for IGF-II over IGF-I. Glycosylation of IGFBP-6 may affect its secretion, in vivo stability or localization, but does not affect its ligand binding properties.     

High-yield bacterial expression and structural characterization of recombinant human insulin-like growth factor binding protein-2

The diverse biological activities of the insulin-like growth factors (IGF-1 and IGF-2) are mediated by the IGF-1 receptor (IGF-1R). These actions are modulated by a family of six IGF-binding proteins (IGFBP-1-6; 22-31 kDa) that via high affinity binding to the IGFs (K_D ∼ 300-700 pM) both protect the IGFs in the circulation and attenuate IGF action by blocking their receptor access. In recent years, IGFBPs have been implicated in a variety of cancers. However, the structural basis of their interaction with IGFs and/or other proteins is not completely understood. A critical challenge in the structural characterization of full-length IGFBPs has been the difficulty in expressing these proteins at levels suitable for NMR/X-ray crystallography analysis. Here we describe the high-yield expression of full-length recombinant human IGFBP-2 (rhIGFBP-2) in Escherichia coli. Using a single step purification protocol, rhIGFBP-2 was obtained with >95% purity and structurally characterized using NMR spectroscopy. The protein was found to exist as a monomer at the high concentrations required for structural studies and to exist in a single conformation exhibiting a unique intra-molecular disulfide-bonding pattern. The protein retained full biologic activity. This study represents the first high-yield expression of wild-type recombinant human IGFBP-2 in E. coli and first structural characterization of a full-length IGFBP.     

Influence of dietary protein on insulin-like growth factor binding proteins in the chicken

We determined the effect of dietary protein on the distribution of insulin-like growth factor (IGF) binding proteins in chicken plasma. Three groups of male broilers (n=6 per group) were fed (ad libitum) isocaloric diets containing 12, 21 or 30% dietary protein. Birds were fed respective diets beginning at 7 days of age and killed at 28 days. No differences were observed between adequate (21%) and high (30%) protein intakes for any of the parameters investigated (growth criteria, plasma levels of IGF-I, growth hormone or IGF-binding proteins). Feeding protein deficient diets (12%) resulted in a 34% decrease in body weight, 17% decrease in feed intake and a 39% increase in feed/gain ratio. IGF-binding proteins in plasma samples were separated by SDS-PAGE and transferred to nitrocellulose sheets. Nitrocellulose blots were probed with [¹²⁵I]chicken IGF-II. Four regions of binding activity corresponding to 70, 43, 30 and 24 kDa were observed in all samples. Birds consuming 12% dietary group protein had less than 50% of the 43-kDa binding activity of birds consuming 21 or 30% dietary protein. The 30-kDa binding activity was 42% lower in the 12% dietary protein group compared to birds consuming adequate protein. In contrast, 70- and 24-kDa binding activities were not influenced by dietary protein. Chickens consuming 12% dietary protein had higher levels of growth hormone and lower levels of IGF-I than those consuming 21 or 30% dietary protein. These data indicate that in chickens, the circulating levels of at least two independent IGF-binding proteins are influenced by dietary protein.     

Differential dissociation kinetics explain the binding preference of insulin-like growth factor binding protein-6 for insulin-like growth factor-II over insulin-like growth factor-I

Insulin-like growth factor binding protein-6 binds insulin-like growth factor-II with a marked preferential affinity over insulin-like growth factor-I. The kinetic basis of this binding preference was studied using surface plasmon resonance. Binding of insulin-like growth factor-I and insulin-like growth factor-II to immobilized insulin-like growth factor binding protein-6 fitted a two-site binding kinetic model. Insulin-like growth factor-I and insulin-like growth factor-II association rates were similar whereas the dissociation rate was approximately 60-fold lower for insulin-like growth factor-II, resulting in a higher equilibrium binding affinity for insulin-like growth factor-II. The equilibrium binding affinities of a series of insulin-like growth factor-II mutants were also explained by differential dissociation kinetics. O-glycosylation had a small effect on the association kinetics of insulin-like growth factor binding protein-6. The insulin-like growth factor binding properties of insulin-like growth factor binding protein-6 are explained by differential dissociation kinetics.     

Regulation of insulin-like growth factor binding protein-1 expression during aging

Insulin-like growth factor (IGF) binding protein-1 (IGFBP-1) is primarily produced in the liver during inflammation and regulates biological activities of IGF-I. Here we demonstrate that interleukin-1beta (IL-1beta) stimulates IGFBP-1 mRNA production in a dose-dependent manner in hepatocytes from Fisher 344 rats. Employment of c-Jun N-terminal kinase (JNK) inhibitor SP600125 resulted in 3-fold reduction of IGFBP-1 mRNA and protein levels, indicating that IL-1beta-induced IGFBP-1 production is mediated through JNK activation. We further show that hepatocytes from aged rats (20-22 mo), as compared to young (3-4 mo), exhibit up to 2-fold higher levels of IGFBP-1 in response to IL-1beta. IL-1beta-induced phosphorylation of JNK was also significantly higher in aged hepatocytes, and SP600125 treatment eliminated age-related differences in IGFBP-1 mRNA production. Moreover, glutathione depletion in hepatocytes from young rats potently activated JNK, as well as increased IL-1beta-induced IGFBP-1 mRNA levels, suggesting that age-related oxidative stress underlies the upregulated JNK activation and IGFBP-1 expression.     

Interaction of insulin-like growth factor binding protein-3 with latent transforming growth factor-beta binding protein-1

Insulin-like growth factor binding protein-3 (IGFBP-3) inhibits the replication and promotes apoptosis in various cell lines in an IGF-independent manner. We utilized a yeast two-hybrid system to identify binding partners for IGFBP-3 in a mouse embryo cDNA library. A partial cDNA encoding mouse latent transforming growth factor beta (TGF-) binding protein-1 (LTBP-1) was identified. This cDNA encoded a mouse LTBP-1 mRNA fragment corresponding to amino acid residues 1160–1712. Analysis of C-terminal deleted mutants indicated that the IGFBP-3 interacting domain resides in the 552 residue C-terminal fragment encoding amino acids 831–1383. The interaction of IGFBP-3 with recombinant human LTBP-1 immobilized on nitrocellulose was also demonstrated. Neither binding of IGF-I to IGFBP-3 nor binding of latency associated protein (LAP) with LTBP-1 inhibited the interaction of IGFBP-3 with LTBP-1. Furthermore the large latent complex, ¹²⁵I-TGF-/LAP/LTBP-1 was able to bind to immobilized IGFBP-3. These data demonstrate that IGFBP-3 can bind to LTBP-1 and provide a potential mechanism whereby IGFBP-3 can interact with the TGF- system.     

重组蛋白质纯化技术

90年代以来 ,基因重组技术得到很大的发展 ,基因工程产品的分离纯化的成本约占其全部成本的 6 0 %～ 80 %,因此重组蛋白的分离纯化技术越来越重要。着重介绍了扩张柱床吸附层析技术 ,径向膜层析技术 ,灌注层析技术 ,液液萃取技术 ,置换层析技术和金属螯合亲和层析技术近年来进展情况以及它们的优缺点和应用范围。     

Cloning and characterization of bovine insulin-like growth factor binding protein-3 (bIGFBP-3).

We report for the first time the isolation of a cDNA encoding the complete amino acid sequence for bovine growth hormone-dependent insulin-like growth factor binding protein-3 (bIGFBP-3). The deduced amino acid sequence from the cDNA revealed a mature polypeptide consisting of 264 amino acids and a 27 amino acid putative signal peptide. The amino acid sequence is over 80% homologous with human IGFBP-3 with complete conservation of the 18 cysteine residues and the 3 Asn-linked glycosylation sites. Between the two species there are 44 amino acid substitutions. Northern analysis of the bIGFBP-3 mRNA in bovine tissue revealed a single mRNA species of 1.65 kilobases.     

重组类胰岛素样生长因子-Ⅰ的纯化与复性

目的 获得高纯度和高活性的胰岛素样生长因子（Insulin-like growth factor, IGF-1）;方法 构建好的BL21大肠杆菌工程菌经IPTG诱导,以融合一段截短型半乳糖苷酶及His-tag形式表达IGF-1融合蛋白(约15,000Da),超声破碎,提取包涵体经镍柱亲和层析后, 用羟氨切割纯化的融合蛋白,纯化后的蛋白质在小分子保护剂及GSH/GSSG的存在下复性。结果 经Ni2＋柱亲和层析, IGF-1纯度达90％以上,复性后得到有较高生物活性的IGF-1。结论 IGF-1发酵及纯化和复性方法的建立为大量生产IGF-1打下了基础。     

Transferrin binds insulin-like growth factors and affects binding properties of insulin-like growth factor binding protein-3.

In the circulation, most of the insulin-like growth factors (IGFs) are bound to a ternary 150 kDa complex with IGF-binding protein (IGFBP)-3 and the acid labile subunit. In the current study, we identify transferrin (Tf) by mass spectrometry, and immunoprecipitation as a component of a major IGF-binding fraction separated from human plasma. IGF ligand blotting, cross-linkage experiments and surface plasmon resonance spectrometry have been used to demonstrate the capability of Tf to bind IGFs specifically. In combination with Tf, IGFBP-3 showed a 5-fold higher affinity for IGF-II than IGFBP-3 alone. The data suggest that Tf may play an important role in regulating IGF/IGFBP-3 functions.     

Purification and characterization of human adrenomedullin binding protein-1

We recently discovered that the vascular responsiveness to adrenomedullin (AM), a potent vasoactive peptide, decreased during sepsis and hemorrhage in the rat and was markedly improved by its novel binding protein (AMBP-1). Moreover, AM/AMBP-1 appears to be one of the leading candidates for further development to treat sepsis and hemorrhage. However, the extremely high cost of commercial AMBP-1 limits the development of human AM and AMBP-1 as therapeutic agents. The purpose of this study was to isolate and purify AMBP-1 from normal human serum and test its stability and biological activity under in vitro and in vivo conditions. AMBP-1 was isolated and purified from normal human serum with a yield of about 3.0 mg per 100 mL and purity of >99%. The purified AMBP-1 has a AM-binding capacity similar to that of the commercial AMBP-1. Human AM and human AMBP-1 in combination significantly inhibited lipopolysaccharide-induced tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 production from macrophages. The biological activity of the purified human AMBP-1 was well preserved when stored at 45 degrees C for 5 d in solution or at 100 degrees C for 1 h in powder. Moreover, administration of AM and purified AMBP-1 to hemorrhaged rats attenuated tissue injury and neutrophil accumulation. Purified AMBP-1 in combination with AM also suppressed the hemorrhage-induced rise in serum cytokines TNF-alpha and IL-6. Thus, we have successfully purified biologically active AMBP-1 from human normal serum and demonstrated the stability of purified human AMBP-1. This technique will enable us to further develop human AM/AMBP-1 as a novel treatment for safe and effective therapy of patients with hemorrhagic shock, sepsis, and ischemic injury.     

Purification and characterization of an insulin-like growth factor II variant from human plasma

An insulin-like growth factor II variant (IGF-II variant) was purified from Cohn fraction IV1 of human plasma by ion exchange, gel filtration, and reversed-phase high pressure liquid chromatography. The amino-terminal sequence of the first 35 amino acid residues showed a replacement of Ser-29 of IGF-II with the tetrapeptide Arg-Leu-Pro-Gly of IGF-II variant. Peptides isolated and sequenced after digestion with endoproteinase Asp-N and endoproteinase Glu-C disclosed no differences with the sequence predicted from an IGF-II variant cDNA clone isolated by Jansen, M., van Shaik, F. M. A., van Tol, H., Van den Brande, J. L., and Sussenbach, J. S. (1985) FEBS Lett., 179, 243-246. The molecular ion of intact IGF-II variant was 7809.4 mass units, as measured by plasma desorption mass spectrometry. This is in close agreement with the molecular ion of 7812.8 mass units calculated from the determined sequence and indicates the entire amino acid sequence had been accounted for. Binding of IGF-II variant to purified insulin-like growth factor I (IGF-I) receptors demonstrated a 2-3-fold lower affinity for this receptor compared with IGF-I or IGF-II. The dissociation constants for IGF-I, IGF-II, and IGF-II variant are 0.23, 0.38, and 0.80 nM, respectively. In a growth assay, the concentration of IGF-II and IGF-II variant required to stimulate the half-maximal growth of MCF-7 cells was 4 and 13 nM, respectively. Finally, the amount of IGF-II variant that can be purified by this method constitutes approximately 25% of the total IGF-II isolated from Cohn fraction IV1 of human plasma.