Expression of human epidermal growth factor gene in cyanobacteria

The human epidermal growth factor (hEGF) is a small single-chain polypeptide of 53 amino acid residues. It can stimulate the proliferation of many cell types, mainly those of epidermal and epithelial tissues both in vivo and in vitro. A vector pRL-hEGF was constructed using plasmids pRL-489 and pUC-hEGF. The synthetic hEGF gene was recombined into the downstream of strong promoter psbA in plasmids pRL-489. Then, the vector was introduced into Synechococcus sp. PCC 7002 and Anabaena sp. PCC 7120 by triparental conjugative transfer. The transformation was confirmed by PCR amplification. The pRL-hEGF is thought to be retained as a plasmid form in the transgenic Anabaena sp. PCC 7120, since it can be recovered. However, it has been integrated into the chromosome of Synechococcus sp. PCC 7002 as there is no duplication origin in the pRL-hEGF in this cyanobacterium. and plasmid cannot be isolated from the Synechococcus sp. PCC 7002 either. The radioimmunoassay (RIA) proved that the hEGF gene has been expressed as the protein existed in these two strains of transgenic cyanobacteria, and the hEGF protein in Anabaena sp. PCC 7002 could be secreted into the medium.

Construction of shuttle, expression vector of human tumor necrosis factor alpha (hTNF-alpha) gene and its expression in a cyanobacterium, Anabaena sp. PCC 7120

The construction of the shuttle, expression vector of human tumor necrosis factor alpha (hTNF-alpha) gene and its expression in a cyanobacterium Anabaena sp. PCC 7120 was reported. The 700-bp hTNF cDNA fragments have been recovered from plasmid pRL-rhTNF, then inserted downstream of the promoter PpsbA in the plasmid pRL439. The resultant intermediary plasmid pRL-TC has further been combined with the shuttle vector pDC-8 to get the shuttle, expression vector pDC-TNF. The expression of the rhTNF gene in Escherichia coil has been analyzed by SDS-PAGE and thin-layer scanning, and the results show that the expressed TNF protein with these two vectors is 16.9 percent (pRL-TC) and 15.0 percent (pDC-TNF) of the total proteins in the cells, respectively, while the expression level of TNF gene in plasmid pRL-rhTNF is only 11.8 percent. Combined with the participation of the conjugal and helper plasmids, pDC-TNF has been introduced into Anabaena sg PCC 7120 by triparental conjugative transfer, and the stable transgenic strains have been obtained. The existence of the introduced plasmid pDC-TNF in recombinant cyanobacterial cells has been demonstrated by the results of the agarose electrophoresis with the extracted plasmid samples and Southern blotting with alpha-(32)p labeled hTNF cDNA probes, while the expression of the hTNF gene in Anabaena sp. PCC 7120 has been confirmed by the results of Western blotting with extracted protein samples and human TNF-alpha monoclonal antibodies. The cytotoxicity assays using the mouse cancer cell line L929 proved the cytotoxicity of the TNF in the crude extracts from the transgenic cyanobacterium Anabaena sp. PCC 7120.

AiC1qDC-1, a novel gC1q-domain-containing protein from bay scallop Argopecten irradians with fungi agglutinating activity

The globular C1q-domain-containing (C1qDC) proteins are a family of versatile pattern recognition receptors via their globular C1q (gC1q) domain to bind various ligands including several PAMPs on pathogens. In this study, a new gC1q-domain-containing protein (AiC1qDC-1) gene was cloned from Argopecten irradians by rapid amplification of cDNA ends (RACE) approaches and expressed sequence tag (EST) analysis. The full-length cDNA of AiC1qDC-1 was composed of 733 bp, encoding a signal peptide of 19 residues and a typical gC1q domain of 137 residues containing all eight invariant amino acids in human C1qDC proteins and seven aromatic residues essential for effective packing of the hydrophobic core of AiC1qDC-1. The gC1q domain of AiC1qDC-1, which possessed the typical 10-stranded beta-sandwich fold with a jelly-roll topology common to all C1q family members, showed high homology not only to those of Cl qDC proteins in mollusk but also to those of C1qDC proteins in human. The AiC1qDC-1 transcripts were mainly detected in the tissue of hepatopancreas and also marginally detectable in adductor, heart, mantle, gill and hemocytes by fluorescent quantitative real-time PCR. In the microbial challenge experiment, there was a significant up-regulation in the relative expression level of AiC1qDC-1 in hepatopancreas and hemocytes of the scallops challenged by fungi Pichia pastoris GS115, Gram-positive bacteria Micrococcus luteus and Gram-negative bacteria Listonella anguillarum. The recombinant AiC1qDC-1 (rAiC1qDC-1) protein displayed no obvious agglutination against M. luteus and L. anguillarum, but it aggregated P. pastoris remarkably. This agglutination could be inhibited by D-mannose and PGN but not by LPS, glucan or D-galactose. These results indicated that AiC1qDC-1 functioned as a pattern recognition receptor in the immune defense of scallops against pathogens and provided clues for illuminating the evolution of the complement classical pathway. (C) 2010 Elsevier Ltd. All rights reserved.

The cDNA cloning and mRNA expression of heat shock protein 70 gene in the haemocytes of bay scallop (Argopecten irradians, Lamarck 1819) responding to bacteria challenge and naphthalin stress

Heat shock protein 70 (HSP70) is an important member of the heat shock protein superfamily, and it plays a key role in the process of protecting cells, facilitating the folding of nascent peptides and responding to stress. The cDNA of bay scallop Argopecten irradians HSP70 (designated AIHSP70) was cloned by the techniques of homological cloning and rapid amplification of cDNA end (RACE). The full length of AIHSP70 cDNA was 2651 bp in length, having a 5' untranslated region (UTR) of 96 bp, a 3' UTR of 575 bp, and an open reading frame (ORF) of 1980 bp encoding a polypeptide of 659 amino acids with an estimated molecular mass of 71.80 kDa and an estimated isoelectric point of 5.26. BLAST analysis revealed that the AIHSP70 gene shared high identity with other known HSP70 genes. Three classical HSP signature motifs were detected in AIHSP70 by InterPro, analysis. 3-D structural prediction of AIHSP70 showed that its N terminal ATPase activity domain and,C terminal substrate-binding domain shared high similarity with that in human heat shock protein 70. The results indicated that the AIHSP70 was a member of the heat shock protein 70 family. A semi-quantitive RT-PCR method was used to analyse the expression of AIHSP70 gene after the treatment of naphthalin which is one kind of polycyclic aromatic hydrocarbon (PAH) and the challenge of bacteria. mRNA expression of AIHSP70 in scallop was up-regulated significantly after the stimulation of naphthalin and increased with increasing naphthalin concentration. A clearly time-dependent expression pattern of AIHSP70 was observed after the scallops were infected by Vibrio anguillarum, and the mRNA expression reached a maximum level at 8 h and lasted to 16 h, and then dropped progressively. The results indicated that AIHSP70 could play an important role in mediating the environmental stress and immune response in scallop. (c) 2006 Elsevier Ltd. All rights reserved.

Protein A tangential flow affinity membrane cartridge for extracorporeal immunoadsorption therapy

Tangential flow affinity membrane cartridge (TFAMC) fs a new model of immunoadsorption therapy for hemoperfusion. Recombinant Protein A was immobilized on the membrane cartridge through Schiff base formation for extracorporeal IgG and immune complex removal from blood. Flow characteristics, immunoadsorption capacity and biocompatibility of protein A TFAMC were studied. The results showed that the pressure drop increased with the increasing flow rate of water, plasma and blood, demonstrating reliable strength of membrane at high now rare. The adsorption capacities of protein A TFAMC for IgG from human plasma and blood were measured. The cartridge with 139 mg protein A immobilized on the matrix (6 mg protein A/g dry matrix) adsorbed 553 mg IgG (23.8 mg IgG/g dry matrix) from human plasma and 499.4 mg IgG (21.5 mg IgG/g dry matrix) from human blood, respectively. The circulation time had a major influence on IgG adsorption capacity, but the flow rate had little influence. Experiments in vitro and in vivo confirmed that protein A TFAMC mainly adsorbed Ige and Little of other plasma proteins, and that blood cell damage was negligible. The extracorporeal circulation system is safe and reliable. Copyright (C) 1999 John Wiley & Sons, Ltd.

Capillary electrophoresis study of human serum albumin binding to basic drugs

The applicability of capillary electrophoresis/frontal analysis (CE/FA) for determining the binding constants of the drugs propranolol (PRO) and verapamil (VER) to human serum albumin (HSA) was investigated. After direct hydrodynamic injection of a drug-HAS mixture solution into a coated capillary (32 cm x 50 mu m i.d.), the basic drug was eluted as a zonal peak with a plateau region under condition of phosphate buffer (pH 7.4; ionic strength 0.17) at 12 kV positive running voltage. The unbound drug concentrations measured from the plateau peak heights had good correlation coefficients, r > 0.999. Employing the Scatchard plot, the Klotz plot and nonlinear regression, the drug protein binding parameters, the binding constant and the number of binding sites on one protein molecule, were obtained. The binding constant obtained was compared to a reported equilibrium dialysis result and they are basically in good agreement.

Application of liquid pre-column capillary electrophoresis technique to the study of interaction between drug enantiomers and human serum albumin

Based on the chiral separation of several basic drugs, dimetindene, tetryzoline, theodrenaline and verapamil, the liquid pre-column capillary electrophoresis (LPC-CE) technique was established. It was used to determine free concentrations of drug enantiomers in mixed solutions with human serum albumin (HSA). To prevent HSA entering the CE chiral separation zone, the mobility differences between HSA and drugs under a specific pH condition were employed in the LPC. Thus, the detection confusion caused by protein was totally avoided. Further study of binding constants determination and protein binding competitions was carried out. The study proves that the LPC technique could be used for complex media, particularly the matrix of protein coexisting with a variety of drugs.

Human plasma proteome analysis by multidimensional chromatography prefractionation and linear ion trap mass spectrometry identification

A resurgence of interest in the human plasma proteome has occurred in recent years because it holds great promise of revolution in disease diagnosis and therapeutic monitoring. As one of the most powerful separation techniques, multidimensional liquid chromatography has attracted extensive attention, but most published works have focused on the fractionation of tryptic peptides. In this study, proteins from human plasma were prefractionated by online sequential strong cation exchange chromatography and reversed-phase chromatography. The resulting 30 samples were individually digested by trypsin, and analyzed by capillary reversed-phase liquid chromatography coupled with linear ion trap mass spectrometry. After meeting stringent criteria, a total of 1292 distinct proteins were successfully identified in our work, among which, some proteins known to be present in serum in < 10 ng/mL were detected. Compared with other works in published literatures, this analysis offered a more full-scale list of the plasma proteome. Considering our strategy allows high throughput of protein identification in serum, the prefractionation of proteins before MS analysis is a simple and effective method to facilitate human plasma proteome research.

Purification of human tissue prokallikrein excreted from insect cells by liquid chromatography

Tissue kallikrein, generally existing in living bodies as prokallikrein, is a serine proteinase that has proven of great significance to treat hypertension, cardiopathy and nephropathy. Although the extraction of tissue kallikrein from human urine is the most commonly used method to obtain such a protein, not only the yield is very little, but also the procedure is rather complex. Furthermore, the biological safety is uncertain. Therefore, the preparation of such a protein by genetic engineering method, including gene expression, cell culture, separation and purification, is very important. In this paper, a new method to obtain purified tissue prokallikrein excreted from insect cells by liquid chromatography has been proposed. In contrast to the previously published papers, the purification procedure is simplified to only three steps with the final yield of 57% and the purity of 95%, which is not only convenient, but also low-cost and suitable for the large-scale preparation of such a protein. The purified protein is further validated as prokallikrein by high performance liquid chromatography-mass spectrometry and amino acid sequencing. (c) 2005 Elsevier B.V. All rights reserved.

Characterization of interaction property of multicomponents in Chinese Herb with protein by microdialysis combined with HPLC

Interaction of traditional Chinese Herb Rhizoma Chuanxiong and protein was studied by microdialysis coupled with high performance liquid chromatography. Compounds in Rhizoma Chuanxiong, such as ferulic acid, senkyunolide A and 3-butylphthalide, were identified by HPLC, HPLC-MS and UV-vis. Microdialysis recoveries and binding degrees of compounds in Rhizoma Chuanxiong with human serum albumin (HSA) and other human plasma protein were determined: recoveries of microdialysis sampling ranged from 36.7 to 98.4% with R.S.D. below 3.1%; while binding to HSA ranged from 0 to 91.5% (0.3 mM HSA) and from 0 to 93.5% (0.6 mM HSA), respectively. Compared with HSA, most of compounds bound to human blood serum more extensively and the results showed that binding of these compounds in Rhizoma Chuanxiong was influenced by pH. Two compounds were found to bind to HSA and human blood serum. their binding degrees were consistent with ferulic acid and 3-butylphthalide, the active compounds in Rhizoma Chuanoxiong. (c) 2005 Elsevier B.V. All rights reserved.