Cryoelectron microscopy of protein IX-modified adenoviruses suggests a new position for the C terminus of protein IX

Recombinant human adenovirus is a useful gene delivery vector for clinical gene therapy. Minor capsid protein IX of adenovirus has been of recent interest since multiple studies have shown that modifications can be made to its C terminus to alter viral tropism or add molecular tags and/or reporter proteins. We examined the structure of an engineered adenovirus displaying the enhanced green fluorescent protein (EGFP) fused to the C terminus of protein IX. Cryoelectron microscopy and reconstruction localized the C-terminal EGFP fusion between the H2 hexon and the H4 hexon, positioned between adjacent facets, directly above the density previously assigned as protein IIIa. The original assignment of IIIa was based largely on indirect evidence, and the data presented herein support the reassignment of the IIIa density as protein IX.     

Localization of the N-terminus of minor coat protein IIIa in the adenovirus capsid

Minor coat protein IIIa is conserved in all adenoviruses (Ads) and is required for correct viral assembly, but its precise function in capsid organization is unknown. The latest Ad capsid model proposes that IIIa is located underneath the vertex region. To obtain experimental evidence on the location of IIIa and to further define its role, we engineered the IIIa gene to encode heterologous N-terminal peptide extensions. Recombinant Ad variants with IIIa encoding six-histidine (6His) tag, 6His, and FLAG peptides, or with 6His linked to FLAG with a (Gly₄Ser)₃ linker were rescued and analyzed for virus yield, capsid incorporation of heterologous peptides, and capsid stability. Longer extensions could not be rescued. Western blot analysis confirmed that the modified IIIa proteins were expressed in infected cells and incorporated into virions. In the Ad encoding the 6His-linker-FLAG-IIIa gene, the 6His tag was present in light particles, but not in mature virions. Immunoelectron microscopy of this virus showed that the FLAG epitope is not accessible to antibodies on the viral particles. Three-dimensional electron microscopy and difference mapping located the IIIa N-terminal extension beneath the vertex complex, wedged at the interface between the penton base and peripentonal hexons, therefore supporting the latest proposed model. The position of the IIIa N-terminus and its low tolerance for modification provide new clues for understanding the role of this minor coat protein in Ad capsid assembly and disassembly.     

Induction of the synthesis of a 70,000 dalton mammalian heat shock protein by the adenovirus E1A gene product

We have attempted to determine whether any cellular genes are activated as a result of the action of the adenoviral El A gene. The proteins synthesized in uninfected HeLa cells have been compared to those produced in early adenovirus infected cells. At least one protein, absent from uninfected HeLa cells, was synthesized in large amounts following adenovirus infection. This 70 kd protein was not synthesized in cells infected with the E1A mutant d1312, even when the multiplicity of infection with the mutant was such that the only viral gene not expressed was the E1A gene. Thus the induction of the 70 kd protein requires the expression of the viral E1A gene. The 70 kd protein was also induced by heat shock in uninfected cells. The same 70 kd protein is synthesized in 293 cells, a line of human embryonic kidney cells transformed by a fragment of adenovirus DNA. These cells constitutively express the E1A and E1 B genes.     

用重叠PCR合成植物甜蛋白brazzein基因

目的:为在泡盛曲霉(Aspergillus awamori)中进行表达,采用重叠PCR合成了植物甜蛋白brazzein基因。方法:根据非洲热带植物Pentadiplandra brazzeana产生的天然甜蛋白brazzein的氨基酸序列及泡盛曲霉糖化酶基因glaA的密码子偏爱性,设计并化学合成了2对3’-端互补的寡聚核苷酸,通过PCR延伸获得2条末端有部分重叠的双链核苷酸片段,再通过重叠PCR扩增,合成了用于泡盛曲霉表达的植物甜蛋白brazzein基因。结果:将brazzein基因克隆到pMD18-T载体,随机挑取6个重组质粒测序,结果1个重组质粒有连续4个碱基缺失,3个重组质粒各有1个碱基缺失,2个重组质粒携带的brazzein基因核苷酸序列完全正确。结论:合成的brazzein基因大小162 bp,编码54个氨基酸,推断的氨基酸序列与Pentadiplandra brazzeana产生的天然brazzein完全一致,表明植物甜蛋白brazzein基因成功合成。     

Secretion of human serum albumin from Bacillus subtilis.

We have fused the structural gene (hsa) for human serum albumin (HSA) to the expression elements and signal sequence coding region of each of two genes from Bacillus amyloliquefaciens P, an alpha-amylase gene (amyBamP) and a neutral protease gene (nprBamP). Bacillus subtilis strains harboring either of these gene fusions synthesized a protein with the antigenic characteristics and size (68 kilodaltons) of HSA. Results from pulse-labeling studies indicated that the bacterially produced HSA was secreted from cells which had been converted to protoplasts. Results from similar studies with intact cells suggested that the signal sequence was removed from the hybrid protein, providing further evidence that B. subtilis can translocate this foreign protein across the cell membrane. Signal sequence removal was efficient when the level of HSA synthesis was low. However, in strains which synthesized HSA at a high level, signal sequence removal was less efficient.     

Quantitation and characterization of human platelet glycoprotein IIIa by radioimmunoassay

Glycoprotein IIIa was quantitated in human platelets by radioimmunoassay using antisera specific to platelet membranes and purified glycoprotein IIIa. Glycoprotein IIIa and glycoprotein IIb were isolated from washed platelets by Triton X-114 extraction followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radioiodinated glycoprotein IIIa was further purified by affinity chromatography on Lentil lectin-Sepharose 4B. Purified glycoprotein IIb showed little crossreactivity with 125I-labeled glycoprotein IIIa using the anti-platelet membrane or anti-glycoprotein IIIa antisera on a competition inhibition radioimmunoassay. The expression of glycoprotein IIIa epitopes were the same for the purified glycoprotein IIIa and glycoprotein IIIa in Triton X-100 solubilized platelets. A 66 kDa protein derived from glycoprotein IIIa by limited proteolysis of platelet membranes also expressed the same epitopes as intact glycoprotein IIIa. Solubilized platelets contained approximately 16 micrograms of total glycoprotein IIIa antigen per 10(9) cells. The level of glycoprotein IIIa determined by radioimmunoassay in one patient with Glanzmann's thrombasthenia amounted to 6.7% of normal and it was close to the values obtained by other methods.     

PDGF受体结合域与乙肝病毒核心抗原的融合表达

化学合成血小板源性生长因子受体结合域１３肽基因,并与乙肝病毒核心抗原基因５′端融合,序列分析表明化学合成的１３肽基因及融合后基因的阅读框架正确．将融合基因亚克隆于ｔａｃ启动子控制的ｐＥＴ３ａ表达质粒中并于大肠杆菌中表达．表达产物经ＥＬＩＳＡ、ＷｅｓｔｒｅｎＢｌｏｔ鉴定表明,融合蛋白已被表达,其单位分子量与推算值一致．电镜观察证明所表达的融合蛋白能形成颗粒．     

cis-acting elements and a trans-acting factor affecting alternative splicing of adenovirus L1 transcripts.

Expression of the L1 region of adenovirus is temporally regulated by alternative splicing to yield two major RNAs encoding the 52- to 55-kilodalton (52-55K) and IIIa polypeptides. The distal acceptor site (IIIa) is utilized only during the late phase of infection, whereas the proximal site (52-55K) is used at both early and late times. Several parameters that might affect this alternative splicing were tested by using expression vectors carrying the L1 region or mutated versions of it. In the absence of a virus-encoded or -induced factor(s), only the 52-55K acceptor was used. Decreasing the distance between the donor and the IIIa acceptor had no effect. Removal of the 52-55K acceptor induced IIIa splicing slightly, implying competition between the two acceptors. Fusion of the IIIa exon to the 52-55K intron greatly enhanced splicing of the IIIa junction, suggesting that the IIIa exon does not contain sequences that inhibit splicing. Thus, the lack of splicing to the IIIa acceptor in the absence of a virus-encoded or -induced factor(s) is probably due to the absence of a favorable sequence and/or the presence of a negative element 5' of the IIIa splice junction, or both. The presence of several adenovirus gene products, including VA RNAs, the E2A DNA-binding protein, and the products of E1A and E1B genes, did not facilitate use of the IIIa acceptor. In contrast, the simian virus 40 early proteins, probably large T antigen, induced IIIa splicing. This result, together with those of earlier studies, suggest that T antigen plays a role in modulation of alternative RNA splicing.     

The Synthesis of (±)-Azetidine-2-carboxylic Acid and 2-Pyrrolidinone Derivatives

(±)-Azetidine-2-carboxylic acid and 3-substituted-2-pyrrolidinones were synthesized from 2-pyrrolidinone via 3-bromo-2-methoxy-1-pyrroline (IIIa). The bromide (IIIa) was obtained by the bromination of 2-methoxy-l-pyrroline using NBS.     

Morphogenesis of the long tail fibers of bacteriophage T2 involves proteolytic processing of the polypeptide (gene product 37) constituting the distal part of the fiber

Gene 37 of phage T2 codes for a protein that, as a dimer, constitutes the most distal, receptor-recognizing part of its long tail fibers. It was found that, from a plasmid carrying a fragment of gene 37 that lacked a region of the gene encoding 87 CO2H-terminal amino acid residues, a protein was expressed that was slightly larger than that present in the phage. This size difference could not be accounted for. The missing region of gene 37 and also gene 38 (which codes for the auxiliary protein required for dimerization of protein 37) were cloned. Plasmids were constructed with gene 37, or gene 37 together with gene 38, under inducible control. Independent of the presence of the latter gene, a protein was produced that had the same size as protein 37 in the phage. A pulse-chase experiment revealed that a precursor of protein 37 is synthesized and processed such that approximately 120 amino acid residues, most likely CO2H-terminal, are removed. Therefore, the protein produced from the truncated gene was larger because it cannot be processed. This fact also solved an old puzzle: an amber fragment of protein 37 of phage T2 had been found to be larger than the mature protein. The amber codon could be located 24 codons away from the normal stop codon. Obviously, the fragment cannot be processed. The existence of this fragment demonstrates that processing occurs during phage maturation.     

HTLV-III env gene products synthesized in E. coli are recognized by antibodies present in the sera of AIDS patients

The envelope gene of HTLV-III, the retrovirus directly linked to AIDS, encodes a protein of 856 amino acids. Our sequence analysis of the cloned HTLV-III (HXB-3) env gene and its comparison with other isolates reveal significant divergence, especially in the external portion of this protein. A large segment of the env gene (1800 bp) was inserted into the expression vector pEV-vrf3, and a corresponding 68 kd protein, which encompasses both the extracellular and the membrane-associated regions of the native protein, was produced in E. coli. Several smaller polypeptides, which appear to be internal initiation products, were also produced. All 50 AIDS patient sera obtained from different locations in the United States specifically recognized the bacterially synthesized envelope proteins, as judged by Western blots. This suggests that these proteins will be useful for the diagnosis of HTLV-III infection and possibly as a vaccine against AIDS.     

Chemical synthesis and expression in E. coli of a human Val8-calcitonin gene by fusion to a synthetic human interferon-gamma gene

A gene coding for human Val8-calcitonin (Val8-hCT) was synthesized by the solid-phase phosphite approach and fused to a synthetic human immune interferon-gamma (IFN-gamma) gene. The IFN gene was previously shown to be expressed at a very high level in E. coli [(1986) Gene, in press] due to the control of a strong synthetic promoter and strong ribosome binding site. The cells harboring the fused gene produced 100-150 micrograms per l of bacterial suspension of immunoreactive calcitonin in the form of hybrid IFN-gamma-Val8-hCT protein consisting of 140 amino acids. The Val8-hCT can be released from this protein by CNBr treatment.     

Platelet glycoprotein IIb/IIIa receptor antagonists derived from isoxazolidines

A series of isoxazolidines has been synthesized as mimetics of the RGD sequence and was evaluated as antagonists of the platelet glycoprotein IIb/IIIa receptor. These compounds were shown to be highly potent GPIIb/IIIa antagonists, exhibiting submicromolar potencies.     

Identification of PlAl alloantigen domain on a 66 kDa protein derived from glycoprotein IIIa of human platelets

Incubation of platelets with chymotryptin leads to the exposure of fibrinogen receptors and to the appearance of a 66 kDa membrane component on the surface of platelets. Both glycoprotein IIIa (GP IIIa) and a 66 kDa component were precipitated from detergent extracts of solubilized, surface radiolabeled chymotrypsin-treated platelets by human anti-PlAl antisera. Moreover, the presence of the P1A1 antigen was identified on GP IIIa (but not on GP IIb) and on a 66 kDa protein by means of immunoblot procedures using platelet Triton X-114 extracts and these purified proteins. Anti-PlAl antiserum did not recognize GP IIIa on the surface of intact (untreated) platelets nor the 66 kDa protein on the surface of chymotrypsin-treated platelets of PlAl-negative individuals. The present data demonstrate directly that the 66 kDa protein is derived from GP IIIa and contains the PlAl alloantigen.     

通过细胞内的靶向定位大幅度提高外源蛋白在转基因植株的积累水平

通过体外操作,对豇豆胰蛋白酶抑制剂(cpti)基因进行修饰,获得了一个融合蛋白基因(sck).该基因是在cpti基因的基础上,在其5'端添加了信号肽编码序列,在3'端添加了内质网滞留信号编码序列,旨在引导基因转译产物进入细胞内质网,并最终滞留在内质网及其衍生的蛋白体内.用sck基因转化烟草(Nicotiana tabacum L.),对获得的转基因植株进行ELISA检测.结果表明,含有修饰基因的转基因烟草CpTI蛋白含量有明显提高,比转未修饰cpti基因烟草平均高出2倍,最高单株可达4倍以上,同时转基因植株的抗虫性也有了显著的提高.结果表明,采用外源蛋白靶向定位的策略,可大幅度提高外源蛋白在转基因植物细胞内的积累量,在植物基因工程研究中具有广泛的借鉴意义.     

Preparation and characterization of human interleukin-5 expressed in recombinant Escherichia coli.

The gene coding for human interleukin-5 was synthesized and expressed in Escherichia coli under control of a heat-inducible promoter. High-level expression, 10-15% of total cellular protein, was achieved in E. coli. The protein was produced in an insoluble state. A simple extraction, renaturation and purification scheme is described. The recombinant protein was found to be a homodimer, similar to the natural murine-derived protein. Despite the lack of glycosylation, high specific activities were obtained in three 'in vitro' biological assays. Physical characterization of the protein showed it to be mostly alpha-helical, supporting the hypothesis that a conformational similarity exists among certain cytokines.