Chemical synthesis of a gene for human epidermal growth factor urogastrone and its expression in yeast.

Urdea, Mickey S.; Merryweather, James P.; Mullenbach, Guy T.; Coit, Doris; Heberlein, Ulrike; Valenzuela, Pablo; Barr, Philip J.

doi:10.1073/pnas.80.24.7461

Cited by 102 publications

(44 citation statements)

References 37 publications

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“…To randomise the nucleotides flanking the SOD initiator AUG, synthetic oligonucleotides and the SOD cDNA were manipulated in vitro, as described in Methods, so as to produce the molecules represented at the top of Fig 2. The S24 synthetic molecules shown here were made during the same phosphoramidite synthesis (42) which contained all four nucleotides at the positions designated by an X in Fig 2. Silica was withdrawn from the synthesis at the 21mer, 22mer, 23mer and 24mer stages, and DNA prepared in the usual way. This step subsequently resulted in recombinant molecules with an SD-AUG spacing of 6-9 nucleotides.…”

Section: Cloning and Sequence Of The Sod Cdnamentioning

confidence: 99%

Human Cu/Zn superoxide dismutase cDNA: isolation of clones synthesising high levels of active or inactive enzyme from an expression library

Hallewell¹,

Masiarz²,

Najarian³

et al. 1985

Nucl Acids Res

143

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The molecular cloning and nucleotide sequence of the cDNA for human Cu/Zn superoxide dismutase (SOD) is reported. The tacI promoter has been used to direct the synthesis in E. coli of this SOD which is soluble, stable, and of normal specific activity. The N-terminal methionine is removed from this protein. A construction with a ribosome binding site identical to that of the lacz gene 5' of the initiator methionine codon, resulted in low levels of SOD. An in vitro mutagenesis procedure was used to randomise the four nucleotides preceding the initiator methionine codon and the silent third positions of the codons specifying the second and third amino acids. Analysis of a sample of 500 clones showed that ca. 25 clones synthesised 5% or more of soluble cell protein as SOD. The nucleotide sequences of high level expressors showed a predominance of A and T residues in t e variable positions 5' of the initiator methionine codon. An SOD mutant (ala +gln) was discovered during the sequencing and shown to lack dismutation activity. Secondary structure predictions for the 5' regions of the mRNAs from high and low level expressors support the hypothesis that initiation of translation is much reduced if part of the region complementary to 16s rRNA is base paired in a stem structure.

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Section: Cloning and Sequence Of The Sod Cdnamentioning

confidence: 99%

Human Cu/Zn superoxide dismutase cDNA: isolation of clones synthesising high levels of active or inactive enzyme from an expression library

Hallewell¹,

Masiarz²,

Najarian³

et al. 1985

Nucl Acids Res

143

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“…For this reason, there is much interest in structure-function studies of EGF and EGF-like molecules. Several laboratories have cloned genes for human EGF (7)(8)(9), and others have synthesized either polypeptide fragments of EGF (10)(11)(12) or the entire EGF protein (13). With this technology, it may be possible to design and engineer EGF-like molecules with desirable biological activities, such as growth factor antagonists or agonists.…”

mentioning

confidence: 99%

Solution structure of murine epidermal growth factor: determination of the polypeptide backbone chain-fold by nuclear magnetic resonance and distance geometry.

Montelione¹,

Wüthrich²,

Nice³

et al. 1987

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

150

100

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The polypeptide backbone fold in the solution structure of murine epidermal growth factor has been determined by nuclear magnetic resonance spectroscopy and distance geometry calculations. The results are based on nearly complete sequence-specific resonance assignments and on 333 distance and dihedral-angle constraints; these were determined from nuclear Overhauser effect measurements, identification of hydrogen-bonded amide protons, the known locations of disulfide bonds, and backbone vicinal spin-spin coupling constants. The polypeptide chain of the protein is arranged into two distinct domains. The structures of these domains were determined independently in separate calculations and then combined to obtain an overall view of the protein. The backbone fold thus determined includes the regular backbone structure elements that were previously identified using different techniques for the analysis of the nuclear magnetic resonance data. The distance geometry calculations also provided additional details about the conformations of bends and loops and about the twists of the ,8-sheets.Epidermal growth factor (EGF) is a small single-chain protein containing 53 amino acids and three disulfide bonds (1-3). EGF and the homologous a-type transforming growth factor (TGF-a) appear to play an important role in the molecular mechanisms controlling mammalian cell growth, oncogenesis (4, 5), and wound healing (6). For this reason, there is much interest in structure-function studies of EGF and EGF-like molecules. Several laboratories have cloned genes for human EGF (7-9), and others have synthesized either polypeptide fragments of EGF (10-12) or the entire EGF protein (13). With this technology, it may be possible to design and engineer EGF-like molecules with desirable biological activities, such as growth factor antagonists or agonists. Knowledge of the three-dimensional structure will be indispensable for understanding the structural basis of EGF function. No crystal structure of EGF or of a homologous growth factor is presently available. This paper presents an initial description of the three-dimensional structure of murine EGF (mEGF) in aqueous solution determined by 1H NMR.Previous NMR studies of murine (14-18), rat (19), and human (20) EGF include two papers on the use of NMR techniques for obtaining complete sequence-specific proton resonance assignments and determination of the three-dimensional structure in solution (21-23). So far the principal elements of regular backbone structure have been identified for both mEGF (18) and human EGF (20). In the work described here, the structure determination of mEGF was continued by collection of a more extensive set of experimental NMR constraints and the use of distance geometry calculations for the structural analysis of the NMR data. METHODSExperimental. mEGF (type al) from male submaxillary glands was purified as described (24, 25) and characterized as homogeneous by both C18 reversed-phase and Pharmacia Mono-Q anion-exchange HPLC. These homogeneity checks were ...

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“…Since chemical synthesis of hEGF has been achieved by introduction of the gene coding for hEGF into yeast (55) and E. coli (39), we decided to use antibodies (polyclonal and monoclonal) directed against such hEGF to reexamine hEGF distribution of various kinds of human tissues and organs under normal and pathologic status. The preceding paper has reported the use of rabbit polyclonal anti-hEGF antiserum and monoclonal antibody, both raised against hEGF obtained by the synthetic gene method to detect hEGF in normal human salivary glands and their neoplastic lesions (53), and hEGF staining in pleomorphic adenomas have already been reported in detail (36,53,56).…”

Section: Respiratory Systemmentioning

confidence: 99%

“…For generation of antiserum, both highly purified hEGF from human urine (45) and hEGF produced biotechnologically by the synthetic gene method (39,55) have been used as immunogens. A preceding paper has described the details of the method for immunohistochemical detection of hEGF and the localization of hEGF in normal salivary glands as well as in their neoplastic lesions (36,53).…”

mentioning

confidence: 99%

Widespread distribution of human epidermal growth factor(hEGF) in human tissues and organs under normal and tumorous conditions. Immunohistochemical studies with the use of polyclonal and monoclonal antibodies.

Mori

Naito

Tsukitani

et al. 1989

Acta Histochem. Cytochem

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Chemical synthesis of a gene for human epidermal growth factor urogastrone and its expression in yeast.

Cited by 102 publications

References 37 publications

Human Cu/Zn superoxide dismutase cDNA: isolation of clones synthesising high levels of active or inactive enzyme from an expression library

Human Cu/Zn superoxide dismutase cDNA: isolation of clones synthesising high levels of active or inactive enzyme from an expression library

Solution structure of murine epidermal growth factor: determination of the polypeptide backbone chain-fold by nuclear magnetic resonance and distance geometry.

Widespread distribution of human epidermal growth factor(hEGF) in human tissues and organs under normal and tumorous conditions. Immunohistochemical studies with the use of polyclonal and monoclonal antibodies.

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