Expression and purification of functional recombinant human pigment epithelium-derived factor (PEDF) secreted by the yeast Pichia pastoris

Pigment epithelium-derived factor (PEDF) combines neurotrophic, neuroprotective, anti-angiogenic, anti-tumor and neural stem cell self-renewal properties in a single molecule, making this protein a valuable potential therapeutic agent. We herein analyzed the expression of human recombinant full-length PEDF, and its N- and C-terminal regions (amino acids 1-243 and 195-418, respectively) in three mammalian cell lines (HEK-293T, COS-1, and 26HCMsv), and in the yeast Pichia pastoris. The highest production of recombinant PEDF was achieved in P. pastoris which secreted approximately 30 microg of full-length rPEDF, and 47 microg of C-terminal/ml of culture medium. Full-length rPEDF was purified by one-step Ni-chelating high-performance liquid chromatography, recovering almost 70% of secreted rPEDF with a purity of 98.6%. The C-terminal region of PEDF was isolated by low-pressure liquid chromatography, recovering around 4% of the recombinant molecule with a purity of 98%. The N-terminal region of PEDF was not secreted by any expression system assayed. The two isolated recombinant PEDF polypeptides inhibited in vitro endothelial cell migration, and full-length rPEDF also increased cerebellar granule cell survival, thus demonstrating their biological activity. These polypeptides can be used to investigate the therapeutic role of PEDF in cancer, neurodegenerative and ocular diseases, and stem cell-based therapies.     

Codon preference optimization increases heterologous PEDF expression

Pigment epithelium-derived factor (PEDF) is widely known for its neurotrophic and antiangiogenic functions. Efficacy studies of PEDF in animal models are limited because of poor heterologous protein yields. Here, we redesigned the human PEDF gene to preferentially match codon frequencies of E coli without altering the amino acid sequence. Following de novo synthesis, codon optimized PEDF (coPEDF) and the wtPEDF genes were cloned into pET32a containing a 5' thioredoxin sequence (Trx) and the recombinant Trx-coPEDF or Trx-wtPEDF fusion constructs expressed in native and two tRNA augmented E coli hosts - BL21-CodonPlus(DE3)-RIL and BL21-CodonPlus(DE3)-RP, carrying extra copies of tRNAarg,ile,leu and tRNAarg,pro genes, respectively. Trx-PEDF fusion proteins were isolated using Ni-NTA metal affinity chromatography and PEDF purified after cleavage with factor Xα. Protein purity and identity were confirmed by western blot, MALDI-TOF, and UV/CD spectral analyses. Expression of the synthetic gene was ～3.4 fold greater (212.7 mg/g; 62.1 mg/g wet cells) and purified yields ～4 fold greater (41.1 mg/g; 11.3 mg/g wet cell) than wtPEDF in the native host. A small increase in expression of both genes was observed in hosts supplemented with rare tRNA genes compared to the native host but expression of coPEDF was ～3 fold greater than wtPEDF in both native and codon-bias-adjusted E coli strains. ΔGs at -3 to +50 of the Trx site of both fusion genes were -3.9 kcal/mol. Functionally, coPEDF was equally as effective as wtPEDF in reducing oxidative stress, promoting neurite outgrowth, and blocking endothelial tube formation. These findings suggest that while rare tRNA augmentation and mRNA folding energies can significantly contribute to increased protein expression, preferred codon usage, in this case, is advantageous to translational efficiency of biologically active PEDF in E coli. This strategy will undoubtedly fast forward studies to validate therapeutic utility of PEDF in vivo.     

PEDF: a potential molecular therapeutic target with multiple anti-cancer activities

Pigment epithelium-derived factor (PEDF) is an endogenously produced protein that is widely expressed throughout the human body, and exhibits multiple and varied biological activities. Already established as a potent anti-angiogenic molecule, PEDF has recently shown promise as a potential anti-tumour agent, causing both direct and indirect tumour suppression. Here, we explore the unique anti-tumour properties of PEDF and discuss its role as an effective anti-angiogenic, anti-proliferative and pro-differentiation factor. We also discuss the prospects for PEDF therapy and the need for a closer evaluation of issues such as delivery, stability and potential toxicity.     

Pigment epithelium-derived factor (PEDF)-induced apoptosis and inhibition of vascular endothelial growth factor (VEGF) expression in MG63 human osteosarcoma cells

Pigment epithelium-derived factor (PEDF) has been shown to be the most potent inhibitor of angiogenesis in the mammalian eye, thus suggesting that loss of PEDF is involved in angiogenic eye diseases such as proliferative diabetic retinopathy. Angiogenesis is required for tumor growth and progression as well. We, along with others, have recently found that PEDF could inhibit growth of melanoma and hepatocellular carcinoma in nude mice through its anti-angiogenic effects on tumor endothelial cells. However, the possibility of the direct effect of PEDF on tumor cells has remained. In this study, we investigated the effects of PEDF on growth and vascular endothelial growth factor (VEGF) expression in MG63 human cultured osteosarcoma cells. PEDF decreased viable cell number as well as DNA synthesis in MG63 cells in a dose-dependent manner. Furthermore, PEDF was found to increase caspase-3/7 activity and to subsequently induce apoptotic cell death in MG63 cells. PEDF also inhibited VEGF expression in MG63 cells at both mRNA and protein levels. Our present study provides novel beneficial aspects of PEDF on osteosarcoma cells; one is induction of apoptotic cell death of tumor cells, and the other is the suppression of VEGF expression, which would lead to inhibition of tumor angiogenesis. PEDF therefore might be a promising therapeutic agent for treatment of patients with osteosarcoma.     

Pigment epithelium-derived factor (PEDF) shares binding sites in collagen with heparin/heparan sulfate proteoglycans

Pigment epithelium-derived factor (PEDF) is a collagen-binding protein that is abundantly distributed in various tissues, including the eye. It exhibits various biological functions, such as anti-angiogenic, neurotrophic, and neuroprotective activities. PEDF also interacts with extracellular matrix components such as collagen, heparan sulfate proteoglycans (HSPGs), and hyaluronan. The collagen-binding property has been elucidated to be important for the anti-angiogenic activity in vivo (Hosomichi, J., Yasui, N., Koide, T., Soma, K., and Morita, I. (2005) Biochem. Biophys. Res. Commun. 335, 756-761). Here, we investigated the collagen recognition mechanism by PEDF. We first narrowed down candidate PEDF-binding sequences by taking advantage of previously reported structural requirements in collagen. Subsequent searches for PEDF-binding sequences employing synthetic collagen-like peptides resulted in the identification of one of the critical binding sites for PEDF, human α1(I)(929-938) (IKGHRGFSGL). Further analysis revealed that the collagen recognition by PEDF is sequence- and conformation-specific, and the high affinity binding motif is KGXRGFXGL in the triple helix. The PEDF-binding motif significantly overlapped with the heparin/HSPG-binding motif, KGHRG(F/Y). The interaction of PEDF with collagen I was specifically competed with by heparin but not by chondroitin sulfate-C or hyaluronan. The binding sequences for PEDF and heparin/HSPG also overlapped with the covalent cross-linking sites between collagen molecules. These findings imply a functional relationship between PEDF and HSPGs during angiogenesis, and the interaction of these molecules is regulated by collagen modifications.     

The anti-angiogenic factor PEDF is present in the human heart and is regulated by anoxia in cardiac myocytes and fibroblasts

Cardiac diseases such as myocardial infarction and heart failure are among the leading causes of death in western societies. Therapeutic angiogenesis has been suggested as a concept to combat these diseases. The biology of angiogenic factors expressed in the heart such as vascular endothelial growth factor (VEGF) is well studied, whereas data on anti-angiogenic mediators in the heart are scarce. Here we study the expression of the anti-angiogenic factor pigment epithelium-derived factor (PEDF) in the human heart and in human cardiac cells. PEDF expression could be detected in human cardiac tissue on the protein and mRNA levels. PEDF mRNA levels were significantly lower in explanted human ischemic hearts as compared to healthy hearts. Our in vitro experiments showed that human adult cardiac myocytes and fibroblasts constitutively secrete PEDF. In addition to anoxic conditions, cobalt chloride, 2,2'dipyridyl and dimethoxally glycine, which stabilize hypoxia inducible factor-α decreased PEDF expression. Furthermore we show that PEDF inhibits VEGF-induced sprouting. We have identified PEDF in healthy and ischemic human hearts and we show that PEDF expression is down-regulated by low oxygen levels. Therefore, we suggest a role for PEDF in the regulation of angiogenesis in the heart and propose PEDF as a possible therapeutic target in heart disease.     

Upregulated pigment epithelium-derived factor (PEDF) promotes trophoblast apoptosis and inhibits invasion in preeclampsia

Preeclampsia (PE) is a severe pregnancy-specific disorder. Previous findings indicated that pigment epithelium-derived factor (PEDF) was upregulated in placentas of women with PE. Here, we investigated the role of PEDF in trophoblast function, especially under hypoxia. The effects of hypoxia on the morphology of extravillous trophoblast (EVT)-derived HTR-8Svneo cells were observed under inverted microscope. Transfections with Lipofectamine LTX were performed according to the manufacturer's protocol. The expression of PEDF protein and mRNA were confirmed by immunofluorescence (IF) and quantitative real-time PCR (qPCR). Apoptosis was detected by transferase-mediated dUTP nick end labeling (TUNEL) assay, and proliferation of trophoblast was detected by CCK-8 method. The invasion capacity of trophoblast was assessed by Transwell assay. PEDF was expressed in HTR-8/SVneo under both normoxia and hypoxic stress. However, cells of hypoxia groups had higher expression level of PEDF, increased apoptosis and decreased invasion capability, as compared with normoxia group. Moreover, after transfection with plasmid expressing PEDF gene, overexpression of PEDF modulated trophoblast activities. In addition, PEDF expression was negatively associated with invasion while positively correlated with apoptosis.Our data suggest that PEDF is an important factor to maintain the biological function of trophoblast cells, thus representing a rational therapeutic target in PE.     

PEDF 在类风湿关节炎滑膜组织中的表达及临床意义

目的:研究类风湿关节炎患者滑膜组织中色素上皮衍生因子(Piment epithelial-derived factor,PEDF)的表达情况。方法:采用免疫组化法,检测30例类风湿关节炎活动期膝关节滑膜组织中PEDF蛋白表达,以16例退行性关节炎患者、16例正常人及该30例患者治疗后(稳定期)关节滑膜组织中PEDF蛋白作对照,进行对比分析。结果:PEDF在类风湿关节炎患者明显低于正常人、退行性关节炎患者滑膜组织中的表达,在活动期滑膜组织中的表达明显低于稳定期,组间比较,差异均有统计学意义(P均<0.05)。结论:PEDF与类风湿关节炎的疾病过程密切相关,针对色素上皮衍生因子的靶点治疗有望成为类风湿关节炎治疗的新的方向及策略。     

Pigment-Epithelium Derived Factor (PEDF) and the human ovary: A role in the generation of ROS in granulosa cells

Aims

Pigment Epithelium Derived Factor (PEDF) is a multifunctional factor, which was found in mouse ovary and in human ovarian follicular fluid (FF). Its ovarian functions include anti-angiogenic actions. This study aimed to explore other PEDF-actions and the sites of PEDF expression in the human ovary.

Materials and methods

We used paraffin-embedded human ovarian sections for PEDF-immunohistochemistry and IVF-derived human granulosa cells (GCs) for RT-PCR, Western blotting and functional studies, including measurement of cell viability (ATP-assay), apoptosis (caspase-assay) and reactive oxygen species (ROS).

Key findings

Immunohistochemistry revealed PEDF in the cytoplasm of GCs of avascular follicles from the preantral to the antral stage and in FF. PEDF was also found in luteinized GCs of the highly vascularized corpus luteum, a result not in line with a sole anti-angiogenic action. Like GCs in vivo, cultured human luteinizing GCs express PEDF. They also responded to exogenous recombinant PEDF. In low concentrations PEDF did not affect cell viability but caused generation ROS. ROS-induction by PEDF was a concentration-dependent process and may be due to the activity of NADPH oxidase (NOX) type 4 and/or 5, which as we found are expressed by GCs. An antioxidant and apocynin, which inhibits NOX, blocked ROS generation. High levels of exogenous recombinant PEDF induced apoptosis of GCs, which was prevented by antioxidants, implying involvement of ROS.

Significance

PEDF is emerging as an ovarian factor, which has unexpected ROS-augmenting activities in the human ovary. It may be involved in ovarian ROS homeostasis and may contribute to oxidative stress.     

Identification of active sequences in the L4a domain of laminin α5 promoting neurite elongation

Laminin α5 is an extracellular matrix protein containing multiple domains implicated in various biological processes, such as embryogenesis and renal function. In this study, we used recombinant proteins and synthetic peptides to identify amino acid residues within the short arm region of α5 that were critical for neurite outgrowth activity. The short arm of α5 contains three globular domains (LN, L4a, and L4b) and three rodlike elements (LEa, LEb, and LEc). Recombinant proteins comprised of the α5 short arm fused with a Fc tag produced in 293 cells were assayed for PC12 (pheochromocytoma) cell adhesion and neurite outgrowth activities. Although it did not have cell attachment activity, neurite outgrowth was promoted by the recombinant protein. To narrow the region involved in neurite outgrowth activity, two truncated recombinant proteins were produced in 293 cells. A recombinant protein lacking L4a and LEb lost activity. Furthermore, we synthesized 78 partially overlapping peptides representing most of the amino acid sequences of L4a and LEb. Of the peptides, A5-76 [mouse laminin α5 928-939 (TSPDLFRLVFRY) in L4a] exhibited neurite outgrowth activity. Mutagenesis studies showed that Phe(933) and Arg(934) were involved in neurite outgrowth activity. Moreover, inhibition assays using anti-integrin monoclonal antibodies showed that neurite outgrowth on the α5 short arm was partially mediated by integrin α1β1. However, the antibodies to integrin α1 and β1 did not inhibit neurite elongation on the A5-76 peptide. These results suggest that in addition to cellular interactions with the active site in the L4a domain, the binding of integrin α1β1 seems to modulate neurite elongation on the short arm of α5.     

Characterization of PEDF: A multi‐functional serpin family protein

Pigment epithelium‐derived factor (PEDF) is a 50 kDa secreted glycoprotein that belongs to the non‐inhibitory serpin family group. PEDF has been described as a natural angiogenesis inhibitor with neurotrophic and immune‐modulation properties; it balances angiogenesis in the eye and blocks tumor progression. The mechanisms underlying most of these events are not completely clear; however, it appears that PEDF acts via multiple high affinity ligands and cell receptors. In this review article, we will summarize the current knowledge on the biochemical properties of PEDF and its receptors, the multimodal activities of PEDF and finally address the therapeutic potential of PEDF in treating angiogenesis‐, neurodegeneration‐ and inflammation‐related diseases. J. Cell. Biochem. 106: 769–775, 2009. © 2009 Wiley‐Liss, Inc.     

Protein Kinase Cε Actin-binding Site Is Important for Neurite Outgrowth during Neuronal Differentiation

We have previously shown that protein kinase Cepsilon (PKCepsilon) induces neurite outgrowth via its regulatory domain and independently of its kinase activity. This study aimed at identifying mechanisms regulating PKCepsilon-mediated neurite induction. We show an increased association of PKCepsilon to the cytoskeleton during neuronal differentiation. Furthermore, neurite induction by overexpression of full-length PKCepsilon is suppressed if serum is removed from the cultures or if an actin-binding site is deleted from the protein. A peptide corresponding to the PKCepsilon actin-binding site suppresses neurite outgrowth during neuronal differentiation and outgrowth elicited by PKCepsilon overexpression. Neither serum removal, deletion of the actin-binding site, nor introduction of the peptide affects neurite induction by the isolated regulatory domain. Membrane targeting by myristoylation renders full-length PKCepsilon independent of both serum and the actin-binding site, and PKCepsilon colocalized with F-actin at the cortical cytoskeleton during neurite outgrowth. These results demonstrate that the actin-binding site is of importance for signals acting on PKCepsilon in a pathway leading to neurite outgrowth. Localization of PKCepsilon to the plasma membrane and/or the cortical cytoskeleton is conceivably important for its effect on neurite outgrowth.     

Triad1 regulates the expression and distribution of EHD1 contributing to the neurite outgrowth of neurons after spinal cord injury

Traumatic spinal cord injury is a common and severe complication after an accident. As we all know that neurite outgrowth of neurons is difficult after a spinal cord injury. Endosome system is associated with cargoes transportation and contributes in promoting the neuronal capability for neurite outgrowth. EH domain-containing protein 1 (EHD1) transports proteins through the endosome system, especially in the recycling endosomes and regulating the neurite outgrowth. In mammalian cells, the involvement of the ubiquitin-proteasome system in endosomal sorting has been well established. Two RING fingers and a DRIL (double RING finger-linked) 1 (Triad1) plays an important role in membrane trafficking and its mutant results in the wrong accumulation of receptors in endosomes and plasma membrane. In this current study, we reasonably integrated the results of the above research and investigated the regulating function of Triad1 to EHD1 following the spinal cord injury. We characterized the upregulated expression and distribution of Triad1 and EHD1 in the neurons after SCI and declared the interaction between Triad1 with EHD1 both in vitro and in vivo. Triad1 regulated the interaction between itself and the full-length or EH domain of EHD1, which influenced the neurite outgrowth of PC12 cells. Our data delineate a novel interaction between Triad1 and EHD1 that may contribute to the regulation of neurite outgrowth for neurons after the spinal cord injury.     

Hypoxia negatively regulates antimetastatic PEDF in melanoma cells by a hypoxia inducible factor-independent, autophagy dependent mechanism

Pigment epithelium-derived factor (PEDF), a member of the serine protease inhibitor (SERPIN) superfamily, displays a potent antiangiogenic and antimetastatic activity in a broad range of tumor types. Melanocytes and low aggressive melanoma cells secrete high levels of PEDF, while its expression is lost in highly aggressive melanomas. PEDF efficiently abrogates a number of functional properties critical for the acquisition of metastatic ability by melanoma cells, such as neovascularization, proliferation, migration, invasiveness and extravasation. In this study, we identify hypoxia as a relevant negative regulator of PEDF in melanocytes and low aggressive melanoma cells. PEDF was regulated at the protein level. Importantly, although downregulation of PEDF was induced by inhibition of 2-oxoglutarate-dependent dioxygenases, it was independent of the hypoxia inducible factor (HIF), a key mediator of the adaptation to hypoxia. Decreased PEDF protein was not mediated by inhibition of translation through untranslated regions (UTRs) in melanoma cells. Degradation by metalloproteinases, implicated on PEDF degradation in retinal pigment epithelial cells, or by the proteasome, was also excluded as regulatory mechanism in melanoma cells. Instead, we found that degradation by autophagy was critical for PEDF downregulation under hypoxia in human melanoma cells. Our findings show that hypoxic conditions encountered during primary melanoma growth downregulate antiangiogenic and antimetastasic PEDF by a posttranslational mechanism involving degradation by autophagy and could therefore contribute to the acquisition of highly metastatic potential characteristic of aggressive melanoma cells.     

Age-related expression of PEDF/EPC-1 in human endometrial stromal fibroblasts: implications for interactive senescence

Aging is the major risk factor for many cancers, and age-related changes in the tissue microenvironment can facilitate tumor growth. This study uses human endometrial cells to begin to test the hypothesis that age-related changes in pigment epithelium-derived factor/early population doubling cDNA-1 (PEDF/EPC-1) levels create an environment that is more permissive to tumor growth. Endometrial stromal fibroblasts (ESF) are the predominant cell type in the human endometrium and exert regulatory control over the glandular epithelial cells, which are the source of most tumors. As ESF age in vitro, their ability to regulate appropriate growth and differentiation of epithelial cells declines. Endometrial epithelial cells in primary culture expressed relatively low levels of PEDF/EPC-1 mRNA. In contrast, early passage quiescent ESF from adult donors produce higher levels of the 1.5-kb PEDF/EPC-1 mRNA and 50-kDa secreted protein than epithelial cells. As ESF age in vitro the relative abundance of PEDF/EPC-1 mRNA declines, as does the level of PEDF/EPC-1 protein secreted into cell culture medium. Treatment with PEDF/EPC-1 protein had no effect on ESF proliferation but did inhibit anchorage-dependent and anchorage-independent proliferation of endometrial carcinoma cells in a dose- and time-dependent manner. These findings imply that an age-related loss of PEDF/EPC-1 expression by ESF could eliminate a negative regulator of cancer cell growth and, thereby, contribute to the age-related increase in cancer incidence.     

Pigment epithelium-derived factor (PEDF) binds to caveolin-1 and inhibits the pro-inflammatory effects of caveolin-1 in endothelial cells

Pigment epithelium-derived factor (PEDF) exerts atheroprotective effects both in cell culture and animal models through its anti-oxidative and anti-inflammatory properties. Caveolin-1 (Cav), a major protein component of caveolae in endothelial cells (ECs), plays a role in the progression of atherosclerosis. However, effects of PEDF on Cav-exposed ECs remain unknown. In this study, we examined whether and how PEDF could inhibit the Cav-induced inflammatory and thrombogenic reactions in human umbilical vein ECs (HUVECs). Surface plasmon resonance revealed that PEDF bound to Cav at the dissociation constant of 7.36 × 10⁻⁷ M. Further, one of the major Cav-interacting proteins in human serum was identified as PEDF by peptide mass fingerprinting analysis using BIAcore 1000 combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Exogenously added Cav was taken up into the membrane fraction of HUVECs and dose-dependently increased monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1) and plasminogen activator inhibitor-1 (PAI-1) mRNA levels, all of which were blocked by the simultaneous treatment with 10 nM PEDF. Small interfering RNAs directed against Cav decreased endogenous Cav levels and suppressed gene expression of MCP-1, VCAM-1 and PAI-1 in HUVECs. This study indicates that PEDF binds to Cav and could block the inflammatory and thrombogenic reactions in Cav-exposed HUVECs. Our present study suggests that atheroprotective effects of PEDF might be partly ascribed to its Cav-interacting properties.     

Expression of Ndufb11 encoding the neuronal protein 15.6 during neurite outgrowth and development

Neurite outgrowth (e.g. axonal or dendrite outgrowth) of neurons is necessary for the development and functioning of the central nervous system. It is well accepted that the differentiation of neurons and neurite outgrowth involve alterations in gene expression. Furthermore, mitochondria play a role in different aspects of neurite outgrowth. Here we show that the expression of Ndufb11, a gene encoding the mitochondrial protein NP15.6 is decreased in the course of neuronal differentiation. NP15.6 is homologous to the bovine protein ESSS, a component of the mitochondrial complex 1. The homologous human NDUFB11 gene is localized to Xp11.3-Xp11.23, a region associated with neurogenetic disorders. The down-regulation of NP15.6 correlates with neurite outgrowth of PC12 cells induced by nerve growth factor. Furthermore, we analyzed the expression of Ndufb11 in the embryonic and adult mouse.     

PEDF: anti-angiogenic guardian of ocular function

Sight-threatening eye diseases can be caused and exacerbated by the aberrant growth of new blood vessels. Recent work indicates that this neovascularization not only is a response to a rise in the local concentration of molecules that induce such angiogenesis but also requires a fall in the levels of endogenous molecules that inhibit angiogenesis. One of the most potent of these endogenous inhibitors is pigment epithelium-derived factor (PEDF), which serves as a survival factor for neuronal components of the eye as well as an essential inhibitor of the growth of ocular blood vessels. Its anti-angiogenic activity is selective in that it is effective against newly forming vessels but spares existing ones, and it is reversible. The molecular basis for this delicate control of endothelial cells is beginning to be understood and strategies to test the ability of PEDF to ameliorate or prevent vessel damage in the eye are developing rapidly.     

Identification of neurite outgrowth promoting sites on the laminin alpha 3 chain G domain

Laminins are expressed in specific tissues and are involved in various biological activities including promoting cell adhesion, growth, migration, neurite outgrowth, and differentiation. The laminin alpha3 chain is mainly located in the skin and is also expressed in the floor plate of the developing neural tube. Previously, we showed that the human laminin alpha3 chain LG4 module binds to syndecan-2/4, a membrane-associated proteoglycan, and promotes human fibroblast adhesion. Here, we have evaluated the neurite outgrowth activity of the laminin alpha3 chain LG4 and LG5 modules. Three overlapping recombinant proteins, which contained LG4 and/or LG5 modules of the human laminin alpha3 chain, were prepared using a mammalian cell expression system. Two proteins, rec-alpha3LG4-5 and rec-alpha3LG4, promoted cell attachment and neurite outgrowth of rat pheochromocytoma PC12 cells, but rec-alpha3LG5 was inactive. Twenty-two peptides covering the entire LG4 module were synthesized and tested for cell attachment and neurite outgrowth activity to identify active sites of the LG4 module. A3G75 (KNSFMALYLSKG, alpha3 chain 1411-1422) and A3G83 (GNSTISIRAPVY, alpha3 chain 1476-1487) promoted PC12 cell attachment and neurite outgrowth. Additionally, A3G75 and A3G83 inhibited PC12 cell attachment to rec-alpha3LG4. These results suggest that the A3G75 and A3G83 sites are important for PC12 cell attachment and neurite outgrowth in the laminin alpha3 chain LG4 module. We also conjugated the A3G75 and A3G83 peptides on chitosan membranes to test their potential as bio-materials. These peptide-conjugated chitosan membranes were more active for neurite outgrowth than the peptide-coated plates. These results suggest that the A3G75- and A3G83-conjugated chitosan membranes are applicable as bio-medical materials for neural tissue repair and engineering.     

Single-Step Purification and Biological Activity of Human Nerve Growth Factor Produced from Insect Cells

Human nerve growth factor (NGF) was cloned and engineered for expression in a baculovirus-infected Spodoptera frugiperda (SF-9) insect cell system. Culture supernatants contained 2-3 mg/L of recombinant human NGF. The human NGF produced by this system was purified to apparent homogeneity with a single-step affinity chromatography procedure using a high-affinity monoclonal antibody originally raised against murine NGF. The purification procedure yielded 1-2 mg of pure, human NGF per liter of culture supernatant; i.e., approximately 60% recovery of the human NGF originally released into the culture medium. Although the gene transfected into the SF-9 cells coded for pro-NGF, the NGF recovered after purification was greater than 95% fully processed, mature protein. The KD for the affinity of the pure, recombinant human NGF for NGF receptor in PC12 membranes is 0.20 +/- 0.05 nM. Activation of neurite outgrowth in PC12 cells occurs with ED50 values of 85 +/- 20 pM and 9.6 +/- 1.5 pM for a 3-day primary response and a 1-day secondary response, respectively. The pure, recombinant human NGF also stimulates a significant increase in dopamine content of PC12 cells with an ED50 of 5.8 +/- 2.7 pM. These binding and biological activation properties are consistent with values observed using murine NGF purified from submaxillary glands.