The terminal structure plays an important role in the biological activity of cecropin CMIV

Antibacterial peptides have received increasing attention as a new pharmaceutical substance. But the molecular mechanism of lysis is still poorly understood. CMIV gene and mutant CMIV gene in GST fusion system were expressed. After cleaving with different cleavage reagents, the peptide with an excess of N-terminus and with an un-amidated C-terminus stopped the activity while the peptide with an excess Asn at the C-terminus had the activity level the same as natural CMIV. The results showed that the terminal structure of cecropin CMIV played an important role in its biological activity.     

家蚕抗菌肽CMIV基因结构改造及表达产物的研究

参照天然抗菌肽ＣＭＩＶ组分的氨基酸序列,作了近５０％的改动,根据大肠杆菌偏爱的密码子,设计并人工合成了抗菌肽基因片段．将人工合成的抗菌肽类ＣＭＩＶ基因先重组到测序载体ｐＵＣ１１８上,经过序列分析,发现克隆于载体ｐＵＣ１１８上的基因片段与设计的序列完全一致．再将该基因片段重组到表达载体ｐＥＴ２８（ａ）上,抗菌肽以融合蛋白的形式表达．融合蛋白经镍－金属离子胶亲和层析纯化后,再用ＣＮＢｒ裂解,最终产物具有与天然抗菌肽相同的生物学活性     

Preliminary study on the cleavage of fusion protein GST-CMIV with palladium(II) complex

A novel method for post-treatment of gene-engineered proteins is reported. A coden of Cys-His unit is introduced into the N-terminal of cecropin CMIV by using PCR. The gene is expressed in E. coli fused with GST. After purification, the fusion protein is cleaved by [Pd(en)(H2O)2]2+ at the His-Arg bond and the cecropin CMIV with antibacterial activity is obtained. The preliminary results held some promise of success for application of the palladium(II) complex as cleavage agent for the production of peptide drugs from gene-engineering fusion proteins.     

含有Fxa切割位点的抗菌肽X在大肠杆菌中的融合表达

抗菌肽是昆虫体液免疫的重要成分[1,2 ] ,它们的分子量较小 ,具有抗菌、抗病毒和杀伤某些肿瘤细胞的功能 ,而不破坏人体正常细胞。基于它的这种选择性效应和分子小、无抗原性的特点 ,可望成为新一代的抗菌、抗肿瘤药物。然而 ,天然抗菌肽来源十分困难 ,不能满足研究和临床应用的需要 ,通过基因工程技术生产抗菌肽已成为人们普遍关注的焦点。抗菌肽ＣＭＩＶ是从家蚕蛹中分离并测定了其一级结构的新型抗菌肽 ,它由 35个氨基酸组成 ,不含甲硫氨酸 ,Ｃ 末端为酰胺[3 ] 。抗菌肽Ｘ是中国家蚕抗菌肽ＣＭＩＶ的变体 ,其一级结构与天然的抗菌肽ＣＭ…     

Fusion expression of mutated cecropin CMIV in E. coli

A cDNA coding mutated cecropin CMIV from Bombyx mori was synthesized according to its amino acid sequense using E .coli biased codons .The gene was cloned into the fusion expression vector pEZZ318 and was expressed in E .coli HB101.The fusion protein produced was purified by affinity chromatography to yield 26 mg/L fusion product .The anti-bacterial activities of recombinant cecropin CMIV were recovered after cleavage by chemical method.     

Fusion expression of mutated cecropin CMIV in E. coli

A cDNA coding mutated cecropin CMIV fromBombyx mori was synthesized according to its amino acid sequence usingE. coli biased codons. The gene was cloned into the fusion expression vector pEZZ318 and was expressed inE. coli HB101. The fusion protein produced was purified by affinity chromatography to yield 26 mg/L fusion product. The anti-bacterial activities of recombinant cecropin CMIV were recovered after cleavage by chemical method.     

Fusion expression of mutated cecropin CMIV inE. coli

A cDNA coding mutated cecropin CMIV fromBombyx mori was synthesized according to its amino acid sequence usingE. coli biased codons. The gene was cloned into the fusion expression vector pEZZ318 and was expressed inE. coli HB101. The fusion protein produced was purified by affinity chromatography to yield 26 mg/L fusion product. The anti-bacterial activities of recombinant cecropin CMIV were recovered after cleavage by chemical method.     

Biological activity of the Asn-5,Arg-7 tridecapeptide encoded by MF alpha 2 of Saccharomyces cerevisiae.

The precursor predicted by the nucleotide sequence of the MF alpha 2 gene of Saccharomyces cerevisiae contains one copy of the tridecapeptide alpha-factor previously characterized (H2N-Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr-COOH) and one copy of a peptide that contains two conservative amino acid substitutions (H2N-Trp-His-Trp-Leu-Asn-Leu-Arg-Pro-Gly-Gln-Pro-Met-Tyr-COOH). To determine whether the novel molecule possesses biological activity, the Asn-5,Arg-7 tridecapeptide was prepared chemically by solid-phase peptide synthesis. Growth arrest and morphogenesis assays gave identical activity profiles for the Asn-5,Arg-7 peptide and the other gene product, the Gln-5,Lys-7 peptide. The activities of the two peptides were additive and indistinguishable for S. cerevisiae X2180-1A. When present in fourfold molar excess, the biologically inactive desTrp-1,Ala-3 dodecapeptide reversed activity of the Asn-5,Arg-7 and Gln-5,Lys-7 tridecapeptides. Furthermore, neither peptide caused growth arrest of a MATa ste2(Ts) mutant when assayed at the restrictive temperature. These studies suggest that both pheromones interact with the alpha-factor receptor in a similar manner.     

Purified polyoma virus medium T antigen has tyrosine-specific protein kinase activity but no significant phosphatidylinositol kinase activity.

Medium T antigen, the transforming protein of polyoma virus, is associated with pp60c-src and strongly activates its tyrosine-specific protein kinase activity. We investigated whether the medium T-pp60c-src complex is also associated with an activity that phosphorylates the membrane phospholipid phosphatidylinositol, as shown for pp60v-src and p68v-ros, the transforming proteins of Rous sarcoma virus and avian sarcoma virus UR2, respectively. Medium T was purified by affinity chromatography from extracts of polyoma virus-infected mouse fibroblasts. It was bound to antibodies against a peptide corresponding to the carboxy terminus of medium T and released from the immune complex with an excess of the same peptide. In a second step, the partially purified medium T was bound to antibodies against another peptide corresponding to an internal region of medium T and released with excess peptide. Further purification was carried out with a monoclonal antibody against pp60c-src. Samples from each purification step were examined for protein kinase and phosphatidylinositol kinase activity. The highly purified preparations of the medium T-pp60c-src complex showed very low levels of phosphatidylinositol kinase activity, and no difference between medium T from transforming viruses and nontransforming hr-t mutants was detected. In contrast, protein kinase activity was associated with medium T purified from transforming viruses but not from hr-t mutants.     

Site-specific antibodies to the erbB oncogene product immunoprecipitate epidermal growth factor receptor

Antiserum to a defined region (residues 373-383) of the erbB oncogene product immunoprecipitated a 170,000 dalton protein that was phosphorylated in an EGF-sensitive fashion as well as the 125I-EGF-receptor complex from A431 human epidermoid carcinoma cells. Preincubation of the antiserum with an excess of the synthetic peptide corresponding to the defined region blocked the immunoprecipitation of this protein. A partial proteolytic peptide map of this immunoprecipitated 170,000 dalton protein was identical to that of the authentic EGF receptor. These results suggest immunological similarity between the erbB gene product and the EGF receptor.     

A novel function of TDIF-related peptides: promotion of axillary bud formation

Small peptides derived from the CLAVATA3/EMBRYO SURROUNDING REGION-related (CLE) gene family play a key role in various cell-cell communications in land plants. Among them, tracheary element differentiation inhibition factor (TDIF; CLE41/CLE44 peptide) and CLE42 peptide of Arabidopsis have almost identical amino acid sequences and act as inhibitors of tracheary element differentiation. In this study, we report a novel function of TDIF and CLE42. We found by the GUS (β-glucuronidase) reporter gene assay that while CLE41 and CLE44 are expressed preferentially in vascular bundles, CLE42 is expressed strongly in the shoot apical meristem (SAM) and axillary meristems. Overexpression of CLE42 and CLE41 enhanced axillary bud formation in the leaf and cotyledon axils. Before floral transition, the emergence of axillary buds in these plants occurred in an acropetal order. Exogenous supply of either TDIF or CLE42 peptide to the wild type induced similar excess bud emergence. In vascular bundles, the TDIF RECEPTOR (TDR) acts as the main receptor for TDIF. The axillary bud emergence of tdr mutants was little affected by either of the peptides. It was confirmed by scanning electron microscopy that peptide-treated wild-type plants form an axillary meristem-like structure earlier than non-treated plants. SHOOT MERISTEMLESS (STM), a marker gene for meristems, was up-regulated in peptide-treated plants before the axillary meristem becomes morphologically distinguishable. These results indicate that CLE42 peptide and TDIF have an activity to enhance axillary bud formation via the TDR. Judging from its expression pattern, CLE42 may play an important role in the regulation of secondary shoot development.     

Expression of natriuretic peptide receptor mRNA and functional response to atrial natriuretic peptide and C-type natriuretic peptide in rainbow trout (Oncorhynchus mykiss) head kidney leucocytes

The stimulatory effect of vasomodulatory natriuretic peptide hormones on macrophages and peripheral blood leucocytes in mammals is well-established. However, the relationship in lower vertebrates has not been characterised. Expression of atrial natriuretic peptide, ventricular natriuretic peptide and C-type natriuretic peptide-1, and the guanylyl cyclase-linked (GC) natriuretic peptide receptor-A and -B-type receptors (NPR-A and NPR-B, respectively) was determined by PCR from the mRNA of rainbow trout head kidney leucocytes yielding gene fragments with 100% homology to the same respective natriuretic peptide and NPR-A and -B sequences obtained from other rainbow trout tissues. A mixed population of isolated rainbow trout head kidney leucocytes was stimulated in vitro with trout atrial natriuretic peptide (specific NPR-A agonist) and trout C-type natriuretic peptide (NPR-A and -B agonist) as well as the cGMP agonist 8-bromo-cGMP or the GC inhibitor 8-bromo-phenyl-eutheno-cGMP. Respiratory burst was stimulated by trout atrial natriuretic peptide, trout C-type natriuretic peptide-1 and 8-bromo-cGMP in a dose dependant manner with the highest activity as a result of stimulation with trout C-type natriuretic peptide-1 in excess of that achieved by phorbol myristate acetate (PMA). Equimolar concentrations of the inhibitor, inhibited the respiratory burst caused by the natriuretic peptides and 8-bromo-cGMP. The natriuretic peptide receptors on rainbow trout head kidney leucocytes appear to have a stimulatory function with regard to respiratory burst that is activated through a cGMP second messenger pathway and the natriuretic peptides expressed in the head kidney leucocytes may well act in a paracrine/autocrine manner.     

Microbead display by in vitro compartmentalisation: selection for binding using flow cytometry

In vitro compartmentalisation in an emulsion was used to physically link proteins to the DNA that encodes them via microbeads. These microbeads can be selected for catalysis, or, as demonstrated here, for binding. Genes encoding a peptide containing an epitope (haemagglutinin) were enriched to near purity from a 10(6)-fold excess of genes encoding a different peptide by two rounds of selection using flow cytometry, indicating approximately 1000-fold enrichment per round. Single beads can be isolated using flow sorting and the single gene on the bead amplified by polymerase chain reaction. Hence, the entire process can be performed completely in vitro.     

Herpesvirus protease inhibition by dimer disruption

Kaposi's sarcoma-associated herpesvirus (KSHV), like all herpesviruses, encodes a protease (KSHV Pr), which is necessary for the viral lytic cycle. Herpesvirus proteases function as obligate dimers; however, each monomer has an intact, complete active site which does not interact directly with the other monomer across the dimer interface. Protein grafting of an interfacial KSHV Pr alpha-helix onto a small stable protein, avian pancreatic polypeptide, generated a helical 30-amino-acid peptide designed to disrupt the dimerization of KSHV Pr. The chimeric peptide was optimized through protein modeling of the KSHV Pr-peptide complex. Circular dichroism analysis and gel filtration chromatography revealed that the rationally designed peptide adopts a helical conformation and is capable of disrupting KSHV Pr dimerization, respectively. Additionally, the optimized peptide inhibits KSHV Pr activity by 50% at a approximately 200-fold molar excess of peptide to KSHV Pr, and the dissociation constant was estimated to be 300 microM. Mutagenesis of the interfacial residue M197 to a leucine resulted in an inhibitory concentration which was twofold higher for KSHV Pr M197L than for KSHV Pr, in agreement with the model that the dimer interface is involved in peptide binding. These results indicate that the dimer interface, as well as the active sites, of herpesvirus proteases is a viable target for inhibiting enzyme activity.     

The regulatory subunit of a cGMP-regulated protein kinase A of Trypanosoma brucei.

This study reports the identification and characterization of the regulatory subunit, TbRSU, of protein kinase A of the parasitic protozoon Trypanosoma brucei. TbRSU is coded for by a single copy gene. The protein contains an unusually long N-terminal domain, the pseudosubstrate site involved in binding and inactivation of the catalytic subunit, and two C-terminally located, closely spaced cyclic nucleotide binding domains. Immunoprecipitation of TbRSU coprecipitates a protein kinase activity with the characteristics of protein kinase A: it phosphorylates a protein kinase specific substrate, and it is strongly inhibited by a synthetic protein kinase inhibitor peptide. Unexpectedly, this kinase activity could not be stimulated by cAMP, but by cGMP only. Binding studies with recombinant cyclic nucleotide binding domains of TbRSU confirmed that both domains bind cGMP with Kd values in the lower micromolar range, and that up to a 100-fold excess of cAMP does not compete with cGMP binding.     

A synthetic peptide containing loop 4 of nerve growth factor for targeted gene delivery

BACKGROUND: Gene delivery vectors that restrict the expression of a therapeutic gene to a particular type of cells are critical to gene therapy in a complex structure, such as the central nervous system. We constructed a nonviral vector for targeted gene transfer to cells expressing nerve growth factor (NGF) receptor TrkA. METHODS AND RESULTS: The vector was a synthetic chimeric peptide composed of a targeting moiety derived from NGF loop 4 and a DNA-binding moiety of 10 lysine residues. The peptide activated signal transduction pathways of the NGF receptor TrkA in PC12 cells and supported the survival of the cells after serum deprivation. After forming complexes with plasmid DNA, the peptide dose-dependently increased reporter gene expression in PC12 cells, which could be inhibited by excess NGF. The peptide-mediated gene expression was not affected in PC12 cells by co-incubation with a blocking antibody against the low-affinity NGF receptor p75 and was significantly enhanced in NIH3T3 cells stably transfected with TrkA cDNA, suggesting the involvement of the high-affinity NGF receptor TrkA without the participation of p75. Moreover, the peptide did not assist gene transfer in TrkA-poor, but TrkB- and/or TrkC-positive primary cerebellar granule neurons and primary cortical glial cells. CONCLUSIONS: The chimeric peptide reported will be useful in gene delivery to and gene therapy of the nervous system and other tissues/organs with cells expressing TrkA.     

Enhancement of gene transfer using YIGSR analog of Tat-derived peptide

Cell penetrating peptide based gene carriers are notably known for low level of gene transfer. To remedy this, as laminin receptor (LR) has been previously linked to tumor metastasis, the LR-binding domain (YIGSR) as well as a scrambled sequence (SGIYR) were added to Tat-derived peptide sequence (YIGSR-Tat and SGIYR-Tat respectively). Peptides cellular uptake was assessed with high-LR (HT1080) and low-LR (HT29) cell lines by flow cytometry. Their ability to form complexes with DNA was examined using YOPRO-1 fluorescence assay and their transfection efficiencies evaluated using a luciferase reporter gene assay. DNA complexes were formed at (+/-) charge ratios as low as 2:1. While no conclusion could be drawn on the effect of YIGSR sequence on peptides uptake in both cell lines, a significant improvement in gene transfection in HT1080 cells was achieved using YIGSR-Tat compared to Tat and SGIYR-Tat. Additionally this increased efficiency was inhibited by excess free YIGSR. No significant difference in transfection efficiency was observed between Tat, SGIYR-Tat and YIGSR-Tat based complexes in HT29 cells. These studies demonstrate that attachment of receptor-binding ligand (YIGSR) to Tat-derived peptide can improve the efficiency of gene transfer in LR-positive cells (HT1080).     

Immunological characterization of a 36 kDa Fe deficiency specific peptide in barley roots

In a previous paper we reported that an acidic 36 kDa peptide is the most strongly induced peptide among several peptides induced by Fe deficiency in barley roots. In this paper, polyclonal antibodies were raised against the 36 kDa peptide. This peptide appeared in the roots of all the graminaceous species tested (barley, rye, wheat, oat, maize, sorghum and rice) in response to Fe deficiency. More of the peptide was found in the roots of graminaceous species which secrete higher amounts of mugineic acids (MAs) under Fe deficient nutrition status. Induction of the 36 kDa peptide was first observed on the third day of Fe deficiency, rising to a maximum value on the seventh day. The trend has a positive correlation with secretion of MAs during Fe deficiency. Further, resupply of Fe resulted in a decrease in peptide production on the second day, reaching a control level on the seventh day. The rate of decrease in peptide production was observed to be slower than that of MA secretion. Other nutrient stresses such as B excess, B deficiency, Cu excess, Cu deficiency, Mn excess, Mn deficiency, Zn excess and Zn deficiency induced far less of the peptide. The specific expression of the 36 kDa peptide in roots of graminaceous species under Fe deficiency suggested the positive association of the peptide with a specific Fe deficiency tolerance mechanism in graminaceous plants.     

Chemo-enzymatic synthesis and structure-activity study of artificially N-glycosylated eel calcitonin derivatives with a complex type oligosaccharide

Starting from N-glycosylated eel calcitonin derivatives that contain an N-acetyl-D-glucosamine residue specifically at the 3rd, 14th, 20th or 26th amino acid residue, corresponding glycopeptides with a complex-type oligosaccharide attached to the respective amino acid residue were synthesized by means of a transglycosylation reaction catalyzed by an endo-beta-N-acetylglucosaminidase from Mucor hiemalis . The use of a recombinant enzyme and an excess of a glycosyl donor led to a yield in excess of 60%. Calcitonin derivatives containing truncated oligosaccharides were also prepared via digestion of the complex-type N-glycan with exoglycosidases. Using these N-glycosylated calcitonin derivatives, the effect of carbohydrate structure and glycosylation site on the three-dimensional structure and the biological activity of the peptide were studied. The conformation of the peptide backbone did not change irrespective of the carbohydrate structure or the glycosylation site. However, hypocalcemic activity, calcitonin-receptor binding activity and the biodistribution of the derivatives were affected by the glycosylation and were dependent on both the carbohydrate structure and the glycosylation site. Although the larger oligosaccharides tended to hinder receptor binding, the biodistribution altered by N-glycosylation appeared to enhance the hypocalcemic activity in some cases, and the magnitude of the effect was dependent on the site of glycosylation.