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1.
A Kuhn 《Science (New York, N.Y.)》1987,238(4832):1413-1415
The major coat protein of bacteriophage M13 is synthesized as a precursor, the procoat, with a typical leader (signal) sequence of 23 residues at its NH2-terminus. A fusion protein that contains the NH2-terminal 141 residues of cytoplasmic ribulokinase and all but the first ten residues of M13 procoat was made. The fusion protein inserts into the plasma membrane of Escherichia coli and is processed by leader peptidase to give rise to a leader peptide of 155 residues and the mature coat protein of 50 residues. The NH2-terminus of the leader peptide remains in the cytoplasm and is protected from protease added to the medium outside of the cell. This indicates that M13 procoat inserts into the membrane as a loop structure and that the NH2-terminus of a leader peptide remains within the cytoplasm during membrane insertion.  相似文献   

2.
Humoral hypercalcemia of malignancy is a common complication of lung and certain other cancers. The hypercalcemia results from the actions of tumor factors on bone and kidney. We report here the isolation of full-length complementary DNA clones of a putative hypercalcemia factor, and the expression from the cloned DNA of the active protein in mammalian cells. The clones encode a prepro peptide of 36 amino acids and a mature protein of 141 amino acids that has significant homology with parathyroid hormone in the amino-terminal region. This previously unrecognized hormone may be important in normal as well as abnormal calcium metabolism.  相似文献   

3.
利用反转录聚合酶链式反应,结合快速扩增cDNA末端(RACE)技术,克隆了甘薯茎线虫乙酰胆碱酯酶基因cDNA Dd-ace-1.该cDNA序列5′端存在反式剪接引导序列SL1,全长2196bp,包含1个1908bp的开放阅读框(GenBank登录号EF583059);在推导出的635个氨基酸残基的前体蛋白中,N端的前24个氨基酸残基为信号肽,随后的611个氨基酸残基是成熟的乙酰胆碱酯酶序列,其预测的相对分子质量为70144.12.在一级结构中,形成催化活性中心的3个氨基酸残基(Ser223、Glu360和His483)、胆碱结合位点Trp(106)以及在亚基内形成二硫键的6个半胱氨酸完全保守;在电鳐(Torpedo californica)乙酰胆碱酯酶分子的催化功能域中存在14个保守的芳香族氨基酸残基,其中10个在甘薯茎线虫乙酰胆碱酯酶中完全保守.该酶的氨基酸序列与南方根节线虫(Meloidogyne incognita)、胎生网尾线虫(Dictyocaulus viviparous)和秀丽小杆线虫(Caenorhabditis elegans)ACE-1的同源性为68.6%、67.2%和66.3%.与其他线虫乙酰胆碱酯酶的聚类分析显示,该乙酰胆碱酯酶(Dd-ACE-1)与其他线虫乙酰胆碱酯酶ACE-1同属一个支系.  相似文献   

4.
The structure of rat preproatrial natriuretic factor ( preproANF ) was determined by nucleotide sequence analysis of an ANF complementary DNA clone. PreproANF is composed of a hydrophobic leader segment (20 amino acids), a precursor containing one glycosylation site (106 amino acids), and ANF (24 amino acids). Atrial natriuretic factor is located at the carboxyl terminus of the precursor molecule. The human, mouse, and rat genomes each contain a single ANF gene which is highly conserved.  相似文献   

5.
通过RT-PCR从斑点叉尾鮰肝脏中克隆编码hepcidin原前体肽的基因"pIH"。与参考序列相比,显示两个氨基酸残基的变异。通过对编码hepcidin原前体肽的基因添加EcoR I和HindⅢ酶切位点,选择含"trxA"融合头的pET32a(+)作为表达质粒、E.coliBL21(DE3)作为工程菌,成功构建"pET32a-pIH"原核表达系统。阳性克隆经1 mmol.L-1IPTG诱导、在37℃培养12 h后,表达的"trxA-pIH"融合蛋白70%以上可溶。Tricine-SDS-PAGE分析表明,细胞经超声破碎后的上清通过固化金属离子亲和层析(IMAC)纯化后,可获得高纯度的目的蛋白。  相似文献   

6.
Most proteins destined for export from Escherichia coli are made as precursors containing amino-terminal leader sequences that are essential for export and that are removed during the process. The initial step in export of a subset of proteins, which includes maltose-binding protein, is binding of the precursor by the molecular chaperone SecB. This work shows directly that SecB binds with high affinity to unfolded maltose-binding protein but does not specifically recognize and bind the leader. Rather, the leader modulates folding to expose elements in the remainder of the polypeptide that are recognized by SecB.  相似文献   

7.
Basic and acidic fibroblast growth factors (FGF's) are potent mitogens for capillary endothelial cells in vitro, stimulate angiogenesis in vivo, and may participate in tissue repair. An oligonucleotide probe for bovine basic FGF was designed from the nucleotide sequence of the amino-terminal exon of bovine acidic FGF, taking into account the 55 percent amino acid sequence homology between the two factors. With this oligonucleotide probe, a full length complementary DNA for basic FGF was isolated from bovine pituitary. Basic FGF in bovine hypothalamus was shown to be encoded by a single 5.0-kilobase messenger RNA; in a human hepatoma cell line, both 4.6- and 2.2-kilobase basic FGF messenger RNA's were present. Both growth factors seem to be synthesized with short amino-terminal extensions that are not found on the isolated forms for which the amino acid sequences have been determined. Neither basic nor acidic FGF has a classic signal peptide.  相似文献   

8.
根据GenBank中的人溶菌酶(hLYZ)mRNA序列设计引物,以人胎盘组织总RNA为模板,通过RT-PCR方法扩增出长度为0.85 kb的hLYZ cDNA序列,经过序列测定表明所克隆的片段与已发表的hLYZ cDNA序列的同源性为99%。推导的氨基酸序列经blastp分析与人溶菌酶蛋白质前体的同源性为97%,成熟肽的同源性为99%,为进一步构建人溶菌酶基因乳腺特异性表达载体奠定了基础。  相似文献   

9.
The sequence of the entire RNA genome of the type flavivirus, yellow fever virus, has been obtained. Inspection of this sequence reveals a single long open reading frame of 10,233 nucleotides, which could encode a polypeptide of 3411 amino acids. The structural proteins are found within the amino-terminal 780 residues of this polyprotein; the remainder of the open reading frame consists of nonstructural viral polypeptides. This genome organization implies that mature viral proteins are produced by posttranslational cleavage of a polyprotein precursor and has implications for flavivirus RNA replication and for the evolutionary relation of this virus family to other RNA viruses.  相似文献   

10.
Hepcidin是一类分子量较小、富含半胱氨酸的阳离子抗菌肽,TH1-5则是从莫桑比克罗非鱼(Oreochromis mossambicus)中分离到的3种hepcidin cDNA序列中的1种;虽然化学合成的TH1-5的成熟肽显示了对若干细菌的抑菌活性,但通过重组DNA表达的此肽是否也具生物学活性则是未知的。本文参考莫桑比克罗非鱼hepcidin TH1-5的核苷酸序列,以尼罗罗非鱼(Oreochromis niloticus)的肝脏为基因克隆的材料,对其类似于hepcidin TH1-5的成熟肽(mTH)进行了重组DNA表达。在构建的重组表达质粒"pET-32a-mTH"中,mTH基因与携带有6×His-tag标签和肠激酶识别位点的trxA基因融合,25℃下,经1mmol/L IPTG诱导培养8h后,在E.coli BL21(DE3)中成功表达了"trxA-mTH"融合蛋白。经固化金属离子亲和层析(IMAC)纯化后的融合蛋白并不显示抑菌活性,但经肠激酶消化处理后,释放出的重组mTH显示了对革兰氏阳性的单增李斯特菌和金黄色葡萄球菌以及革兰氏阴性的大肠杆菌和铜绿假单胞菌的抑菌活性。  相似文献   

11.
淇河鲫生长激素基因cDNA的克隆和原核高效表达   总被引：1，自引：0，他引：1  
从脑垂体中提取总RNA,用RT-PCR方法扩增并克隆到淇河鲫(Carassius auratus gibelio var)的生长激素(GH)基因cDNA,其GenBank注册号为DQ350437.分析其核苷酸序列和推测的氨基酸序列,结果显示:克隆到的淇河鲫生长激素基因的开放阅读框(ORF)包括633个核苷酸,编码210个氨基酸,其中,包括22个氨基酸的信号肽和188个氨基酸的成熟肽.把GH成熟肽的cDNA克隆入表达载体pET-28a,在大肠杆菌BL21(DE3)表达N端含6个组氨酸的融合多肽.SDS-PAGE结果表明,0.1 mmol/LIPTG诱导表达的蛋白约为23.5 kD,其表达量超过蛋白总量的50%,主要为不溶性的包涵体.细菌裂解液沉淀溶于8mol/L尿素后,用固定化金属配体亲和层析纯化,获得了分子量约为23.5kD的单一蛋白带.  相似文献   

12.
A D-alanine-containing peptide termed dermorphin, with potent opiate-like activity, has been isolated from skin of the frog Phyllomedusa sauvagei. Complementary DNA (cDNA) libraries were constructed from frog skin messenger RNA and screened with a mixture of oligonucleotides that contained the codons complementary to five amino acids of dermorphin. Clones were detected with inserts coding for different dermorphin precursors. The predicted amino acid sequences of these precursors contained homologous repeats of 35 amino acids that included one copy of the heptapeptide dermorphin. In these cloned cDNAs, the alanine codon GCG occurred at the position where D-alanine is present in the end product. This suggests the existence of a novel post-translational reaction for the conversion of an L-amino acid to its D-isomer.  相似文献   

13.
Molecular cloning of the complementary DNA for human tumor necrosis factor   总被引：85，自引：0，他引：85  
Tumor necrosis factor (TNF) is a soluble protein that causes damage to tumor cells but has no effect on normal cells. Human TNF was purified to apparent homogeneity as a 17.3-kilodalton protein from HL-60 leukemia cells and showed cytotoxic and cytostatic activities against various human tumor cell lines. The amino acid sequence was determined for the amino terminal end of the purified protein, and oligodeoxyribonucleotide probes were synthesized on the basis of this sequence. Complementary DNA (cDNA) encoding human TNF was cloned from induced HL-60 messenger RNA and was confirmed by hybrid-selection assay, direct expression in COS-7 cells, and nucleotide sequence analysis. The human TNF cDNA is 1585 base pairs in length and encodes a protein of 233 amino acids. The mature protein begins at residue 77, leaving a long leader sequence of 76 amino acids. Expression of high levels of human TNF in Escherichia coli was accomplished under control of the bacteriophage lambda PL promoter and gene N ribosome binding site.  相似文献   

14.
Human lipoprotein lipase complementary DNA sequence   总被引：60，自引：0，他引：60  
Lipoprotein lipase is a key enzyme of lipid metabolism that acts to hydrolyze triglycerides, providing free fatty acids for cells and affecting the maturation of circulating lipoproteins. It has been proposed that the enzyme plays a role in the development of obesity and atherosclerosis. The human enzyme has been difficult to purify and its protein sequence was heretofore undetermined. A complementary DNA for human lipoprotein lipase that codes for a mature protein of 448 amino acids has now been cloned and sequenced. Analysis of the sequence indicates that human lipoprotein lipase, hepatic lipase, and pancreatic lipase are members of a gene family. Two distinct species of lipoprotein lipase messenger RNA that arise from alternative sites of 3'-terminal polyadenylation were detected in several different tissues.  相似文献   

15.
根据Genebank上登录的鸡的myostatin基因cDNA全长序列以及成熟肽序列设计一对引物,并分别在两引物前设计两个酶切位点EcoRⅠ和KpnⅠ,克隆岭南黄鸡肌肉生长抑制激素的成熟肽蛋白编码基因,然后将特异性片段连接到pMD18-T载体,经酶切、PCR鉴定后,构建了岭南黄鸡真核单纯表达载体pPICZαA-MSTN-m,经测序鉴定,结果表明所克隆的myosta-tin成熟肽基因与Genebank上发表的鸡（AF019621）、猪（AY208121）和家鹅（AF440862）的MSTN-m核酸序列同源性分别可达到99%、92%、86%,但翻译后的成熟蛋白氨基酸序列与鸡、猪和家鹅的同源性可以分别达到100%、99.1%及99.1%。  相似文献   

16.
Li P  Chan HC  He B  So SC  Chung YW  Shang Q  Zhang YD  Zhang YL 《Science (New York, N.Y.)》2001,291(5509):1783-1785
Little is known about the innate defense mechanisms of the male reproductive tract. We cloned a 385-base pair complementary DNA and its genomic DNA named Bin1b that is exclusively expressed in the caput region of the rat epididymis and that is responsible for sperm maturation, storage, and protection. Bin1b exhibits structural characteristics and antimicrobial activity similar to that of cationic antimicrobial peptides, beta-defensins. Bin1b is maximally expressed when the rats are sexually mature and can be up-regulated by inflammation. Bin1b appears to be a natural epididymis-specific antimicrobial peptide that plays a role in reproductive tract host defense and male fertility.  相似文献   

17.
Uromodulin (Tamm-Horsfall glycoprotein): a renal ligand for lymphokines   总被引：18，自引：0，他引：18  
The protein portion of the immunosuppressive glycoprotein uromodulin is identical to the Tamm-Horsfall urinary glycoprotein and is synthesized in the kidney. Evidence that the glycoproteins are the same is based on amino acid sequence identity, immunologic cross-reactivity, and tissue localization to the thick ascending limb of Henle's loop. Nucleic acid sequencing of clones for uromodulin isolated from a complementary DNA bank from human kidney predicts a protein 639 amino acids in length, including a 24--amino acid leader sequence and a cysteine-rich mature protein with eight potential glycosylation sites. Uromodulin and preparations of Tamm-Horsfall glycoprotein bind to recombinant murine interleukin-1 (rIL-1) and human rIL-1 alpha, rIL-1 beta, and recombinant tumor necrosis factor (rTNF). Uromodulin isolated from urine of pregnant women by lectin adherence is more immunosuppressive than material isolated by the original salt-precipitation protocol of Tamm and Horsfall. Immunohistologic studies demonstrate that rIL-1 and rTNF bind to the same area of the human kidney that binds to antiserum specific for uromodulin. Thus, uromodulin (Tamm-Horsfall glycoprotein) may function as a unique renal regulatory glycoprotein that specifically binds to and regulates the circulating activity of a number of potent cytokines, including IL-1 and TNF.  相似文献   

18.
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19.
The molecular basis of the sparse fur mouse mutation   总被引：30，自引：0，他引：30  
The ornithine transcarbamylase-deficient sparse fur mouse is an excellent model to study the most common human urea cycle disorder. The mutation has been well characterized by both biochemical and enzymological methods, but its exact nature has not been revealed. A single base substitution in the complementary DNA for ornithine transcarbamylase from the sparse fur mouse has been identified by means of a combination of two recently described techniques for rapid mutational analysis. This strategy is simpler than conventional complementary DNA library construction, screening, and sequencing, which has often been used to find a new mutation. The ornithine transcarbamylase gene in the sparse fur mouse contains a C to A transversion that alters a histidine residue to an asparagine residue at amino acid 117.  相似文献   

20.
The neuron-specific protein PGP 9.5 is a ubiquitin carboxyl-terminal hydrolase   总被引：42，自引：0，他引：42  
A complementary DNA (cDNA) for ubiquitin carboxyl-terminal hydrolase isozyme L3 was cloned from human B cells. The cDNA encodes a protein of 230 amino acids with a molecular mass of 26.182 daltons. The human protein is very similar to the bovine homolog, with only three amino acids differing in over 100 residues compared. The amino acid sequence deduced from the cDNA was 54% identical to that of the neuron-specific protein PGP 9.5. Purification of bovine PGP 9.5 confirmed that it is also a ubiquitin carboxyl-terminal hydrolase. These results suggest that a family of such related proteins exists and that their expression is tissue-specific.  相似文献   

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