The sequence of human serum albumin cDNA and its expression in E. coli

DNA complementary to human a-fetoprotein (AFP) mRNA was cloned in the plasmid pBR322. Analysis of three overlapping cDNA clones revealed most of the nucleotide sequence of AFP mRNA, and the remaining nucleotides at the 5' end of the mRNA were elucidated from a cloned genomic DNA fragment. The amino acid sequence was deduced from the nucleotide sequence, which revealed 19 amino acids in the signal sequence and 590 amino acids in mature AFP. There are 15 regularly spaced disulfide bridges, which generate a folding structure having three repeating domains. There is one potential N-glycosylation site, Asn-Phe-Thr, in the amino acid sequence. In comparison with mouse AFP, 66% of the amino acid sequence was conserved, with the highest identity (72%) in domain 3, followed by domain 2 (67%) and domain 1 (59%). In comparison with human albumin, a 39% conservation ofprimary structure was found. Again, the similarity was the highest in domain 3 and the lowest in domain 1. Human AFP and human albumin are similar in overall structure, but certain parts of the molecules differ significantly in their predicted secondary structure. a-Fetoprotein (AFP) is a major serum protein (Mr, =70,000) synthesized during fetal life (1-4). Reappearance of AFP in adult serum often signals pathological conditions, particularly hepatocarcinomas and teratocarcinomas (1-4). In contrast, albumin increases steadily during fetal and neonatal growth and shows no apparent changes in concentration associated with development of liver and germ cell tumors.In the past several years, DNAs complementary to mouse (5, 6) and rat (7) AFP mRNAs and rat (8) and human (9, 10) albumin mRNAs have been cloned. Nucleotide sequences of these clones and the amino acid sequences deduced from them have provided valuable information on the structure of these proteins. However, the rat cDNA clones are not full-length and consequently, molecular comparisons have been limited to partial sequences of mouse and rat AFPs. Also, comparisons between AFP and albumin were possible only interspecially because neither mouse albumin nor human AFP mRNA sequences were known in their entirety.In this paper we report the complete nucleotide sequence of human AFP mRNA and the amino acid sequence of AFP. These data allow comparisons of the entire primary structures of AFPs interspecially and AFP and albumin intraspecially. MATERIALS AND METHODSThe production of the cDNA clone, pHAF2, containing 841 nucleotides or about 40% of the human AFP mRNA sequence at the 3' end was described (11). By using the nick-translated insert of pHAF2 as a probe, a second clone, pHAF6, with an additional 320 nucleotides at the 5' end was obtained (Fig. 1).

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“…Comparisons of amino acid sequences between human AFP and human albumin (9,10) are also shown in Fig. 3.…”

Section: Resultsmentioning

confidence: 99%

“…In the past several years, DNAs complementary to mouse (5,6) and rat (7) AFP mRNAs and rat (8) and human (9,10) albumin mRNAs have been cloned. Nucleotide sequences of these clones and the amino acid sequences deduced from them have provided valuable information on the structure of these proteins.…”

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confidence: 99%

Primary structures of human alpha-fetoprotein and its mRNA.

Morinaga¹,

Sakai²,

Wegmann³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

257

149

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“…5Ј-GGACCTTGCCAAGTATATCTGTGA-3Ј (position 921-944, outer sense), 14 5Ј-GGAAGACATCCTTTGCCTCAGCAT-3Ј (position 1103-1126, outer antisense), † Steatosis (%) refers to the absolute number of patients with any steatosis in the PCR-positive and -negative groups.…”

mentioning

confidence: 99%

“…5Ј-TCCAGTAAACTGAAGGAATGCTGT-3Ј (position 961-984, inner sense), 14 5Ј-CAAAATCAGCAGCTAATGAAGGCA-3Ј (position 1052-1075, inner antisense).…”

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confidence: 99%

Intrahepatic Hepatitis C Viral Rna Status of Serum Polymerase Chain Reaction-Negative Individuals With Histological Changes on Liver Biopsy

Barrett

2001

Hepatology

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Hepatitis C virus (HCV) infection is a major public health problem worldwide. 1 Diagnosis of HCV is based on serological detection of anti-HCV antibodies by enzyme-linked immunosorbent assay (ELISA) supplemented with recombinant immunoblot assay (RIBA) and detection of HCV RNA in serum by polymerase chain reaction (PCR), because anti-HCV antibodies alone cannot discriminate active from past infection. 2 Histological assessment is also important in assessing disease status. 3 For individuals testing anti-HCV positive but negative for HCV RNA in serum, the precise diagnosis remains unclear. Whether they have spontaneously resolved past infection or currently have active infection undetectable in serum but which could be detectable in liver, since viral levels have been demonstrated to be 10 4 -fold higher in liver than in serum, is unclear. [4][5][6][7][8] If the latter is true and low level active infection is present, then these individuals represent a group well suited for therapeutic intervention because a low viral load is accepted as a favorable prognostic factor for therapy. 9 Because few studies to date have investigated HCV RNA status in the liver of anti-HCV-positive serum HCV RNA individuals, it is important to assess intrahepatic HCV RNA status in these individuals to determine whether they have truly spontaneously resolved past infection or have current infection with similar clincial, histological, and virological profiles as serum PCR-positive patients. [4][5][6][7] The discovery in 1994 that HCV-contaminated anti-D immunoglobulin had been administered to Rhesus-negative women in 1977 and 1991 to 1993 led to a national screening program conducted by the Irish Blood Transfusion Service Board (BTSB). 10 As a result of this program, a large group of PCR-positive (n ϭ 490) and antibody positive PCR-negative individuals (n ϭ 526) were identified and referred to 6 designated Liver Centers for further evaluation. 10,11 The aim of this study was to assess the significance of histological changes in the liver of serum PCR-negative women who had undergone liver biopsy at this center by detailed investigation of clinical, histological, and intrahepatic HCV RNA status. For the purpose of comparison, some clinical and histological data from a large group of serum PCR-positive women who received HCV-contaminated anti-D over the same time period as the serum PCR-negative women is also presented. PATIENTS AND METHODSPatients. As a result of the national screening program, 100 PCRpositive and 96 PCR-negative women who had received HCV-contaminated anti-D were randomly referred to this center and constitute a representative cross-section of the total infected cohort.Of the 96 PCR-negative women, 42 (43.8%) were still RIBApositive and 6 (6.2%) had elevated ALT levels at the time of presentation. Symptoms, consisting mainly of fatigue and arthralgia dating from the time of inoculation were reported by 69 (71.9%) women. It

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“…In 1975 the amino acid sequences of bovine (Brown, 1975) and human (Behrens et al, 1975;Meloun et al, 1975) albumins were reported. Since that time, the cDNA sequence, and thus the inferred amino acid sequence, has been determined for the following vertebrate albumins: human (Lawn et al, 1981;Dugaiczyk et al, 1982), rat (Sargent et al, 1981b), mouse (Minghetti et al, 1985), pig (Weinstock & Baldwin, 1988), sheep (Brown et al, 1989), frog (Haefliger et al, 1989), and salmon (Byrnes & Gannon, 1990). The most characteristic feature of the albumin sequence is the repetitious pattern of 17 disulfide bridges that organizes the molecule into three homologous, yet distinct, domains.…”

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confidence: 99%

Characterization, primary structure, and evolution of lamprey plasma albumin

Gray

Doolittle

1992

Protein Science

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The most abundant protein found in blood plasma from the sea lamprey (Petromyzon marinus) has the hallmarks of a plasma albumin: namely, high abundance, solubility in distilled water, a small number of tryptophans, and a high content of cysteines and charged residues. As in other vertebrate albumins, not all the cysteines are disulfide bonded. An unusual feature of this protein is its molecular weight of 175,000, roughly 2.5 times the size of other vertebrate albumins. Its amino acid sequence, deduced from a series of overlapping cDNA clones, can be aligned with other members of the gene family including plasma albumin, alpha-fetoprotein, and vitamin-D binding protein, confirming that it is indeed an oversized albumin. An unusual feature of the sequence is a 28-amino acid stretch consisting of a serine-threonine repeat with the general motif (STTT). Lamprey albumin contains a 23-amino acid putative signal peptide and a 6-residue putative propeptide, which, when cleaved, yield a mature protein of 1,394 amino acids with a calculated molecular weight of 157,000. The sequence also includes nine potential N-linked glycosylation sites (Asn-X-Ser/Thr), consistent with observation that lamprey albumin is a glycoprotein. If all the potential glycosylation sites were occupied by clusters of 2,000 molecular weight each, the total molecular weight would be 175,000. Like other members of the gene family, lamprey albumin is composed of a series of 190-amino acid repeats, there being seven such domains all together. Quantitative amino acid sequence comparisons of lamprey albumin with the other members of the gene family indicate that it diverged from an ancestral albumin prior to the gene duplications leading to this diverse group. This notion is confirmed by the pattern of amino acid insertions and deletions observed in a consideration of all domains that compose this family. Furthermore, it suggests that the invention of albumin antedates the vertebrate radiation.Keywords: plasma albumin; protein evolution; sea lamprey Plasma albumin is the most abundant protein in vertebrate blood plasma and also among the most readily purified. The ability to secure relatively pure albumin in large quantities has led to it becoming one of the most familiar and extensively studied of all proteins. Its principal role is thought to be that of a transport protein, reversibly binding fatty acids, bilirubin, and a myriad of other molecules, as well as its being a major contributor to the effective osmotic pressure of the plasma. As a result of its ligand-binding properties and high concentration, albumin provides a stabilizing effect on plasma solute levels, buffering the concentration of metabolites. Paradoxically, humans and some other animals appear to be quite capable of surviving with levels of albumin in

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The sequence of human serum albumin cDNA and its expression in E. coli

Cited by 188 publications

References 35 publications

Primary structures of human alpha-fetoprotein and its mRNA.

Primary structures of human alpha-fetoprotein and its mRNA.

Intrahepatic Hepatitis C Viral Rna Status of Serum Polymerase Chain Reaction-Negative Individuals With Histological Changes on Liver Biopsy

Characterization, primary structure, and evolution of lamprey plasma albumin

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