Chemical synthesis of a gene coding for human angiogenin, its expression in Escherichia coli and conversion of the product into its active form

Denèfle, Patrice; S, Kovarik; Guitton, Jean-Dominique; Cartwright, Terence; Mayaux, Jean‐François

doi:10.1016/0378-1119(87)90158-2

Cited by 56 publications

(30 citation statements)

References 15 publications

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“…Six different preparations of human recombinant ANG (rANG) were produced in Escherichia coli and purified as described (21). They migrated as a single band in SDS/PAGE (22).…”

Section: Methodsmentioning

confidence: 99%

“…rANG differs from natural human angiogenin (nANG) in possessing N-terminal methionine instead of the natural N-terminal pyroglutamic residue (23), but it has been shown to be active as an angiogenic factor in the chorioallantoic membrane assay (21) and to induce blood vessel growth in the rabbit cornea (unpublished data). It exhibits characteristic ribonucleolytic activity (21). nANG was isolated from human plasma as described (2).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Specific binding of angiogenin to calf pulmonary artery endothelial cells.

Badet

Soncin

Guitton

et al. 1989

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

123

106

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Specific binding of angiogenin (ANG) to calf pulmonary artery endothelial cells was demonstrated. Cellular binding at 4TC of 125-Ilabeled human recombinant ANG was time and concentration dependent, reversible, and saturable in the presence of increasing amounts of the unlabeled molecules. The interaction was shown to be specific since a large excess of unlabeled ANG reduced labeled ANG binding by >80%, whereas similar doses of RNase A, a structurally related protein, had no effect. Scatchard analyses of binding data revealed two apparent components. High-affinity sites with an apparent dissociation constant of 5 x 10-9 M were shown to represent cell-specific interactions. The second component, comprising low-affinity/high-capacity sites with an apparent dissociation constant of 0.2 x 10-6 M, was essentially associated with pericellular components. High-affinity ANG binding sites varied with cell density and were found on other endothelial cells from bovine aorta, cornea, and adrenal cortex capillary but not on Chinese hamster lung fibroblasts. Divalent copper, a modulator of angiogenesis, was found to induce a severalfold increase in specific cell-bound radioactivity. Placental ribonuclease inhibitor, a tight-binding inhibitor of both ribonucleolytic and angiogenic activities of ANG, abolished 1251-labeled human recombinant ANG binding only in the absence of copper.

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“…Six different preparations of human recombinant ANG (rANG) were produced in Escherichia coli and purified as described (21). They migrated as a single band in SDS/PAGE (22).…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Specific binding of angiogenin to calf pulmonary artery endothelial cells.

Badet

Soncin

Guitton

et al. 1989

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

123

106

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“…RNase A has been expressed in yeast by secreting the expressed protein (Raines & Rutler, 1989). Angiogenin, which shows strong sequence homology to RNase A and has ribonuclease activity, has been expressed in E. coli under the control of the trp promoter (Denefle et al, 1987;Shapiro et al, 1988) and also has been expressed in baby hamster kidney cells (Kurachi et al, 1988). RNase T1 has been expressed in E. coli by linking the gene to the signal peptide of the omp A protein, so that active ribonuclease is secreted into the periplasmic space (Quaas et al, 1988).…”

Section: Ribonuclease Expressionmentioning

confidence: 99%

Cis proline mutants of ribonuclease A. I. thermal stability

Schultz

Baldwin

1992

Protein Science

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A chemically synthesized gene for ribonuclease A has been expressed in Escherichia coli using a T7 expression system (Studier, F.W., Rosenberg, A.H., Dunn, J.J., & Dubendorff, J.W., 1990, Methods Enzymol. 185, 60-89). The expressed protein, which contains an additional N-terminal methionine residue, has physical and catalytic properties close to those of bovine ribonuclease A. The expressed protein accumulates in inclusion bodies and has scrambled disulfide bonds; the native disulfide bonds are regenerated during purification. Site-directed mutations have been made at each of the two cis proline residues, 93 and 114, and a double mutant has been made. In contrast to results reported for replacement of trans proline residues, replacement of either cis proline is strongly destabilizing. Thermal unfolding experiments on four single mutants give AT,,, e 10 "C and AAGo (apparent) = 2-3 kcal/mol. The reason is that either the substituted amino acid goes in cis, and cis H trans isomerization after unfolding pulls the unfolding equilibrium toward the unfolded state, or else there is a conformational change, which by itself is destabilizing relative to the wild-type conformation, that allows the substituted amino acid to form a trans peptide bond.

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“…maintain E. coli and B. lactofermentum plasmids, respectively. Conditions for the expression of heterologous expression in E. coli have been described previously (7,11). B. lactofermentum RP2 is a spontaneous rifampin-resistant isolate of B. lactofermentum ATCC 21086, the endogenous plasmids of which were cured after extensive protoplastisation and regeneration of the cell wall (27a).…”

mentioning

confidence: 99%

Purification, cloning, and primary structure of a new enantiomer-selective amidase from a Rhodococcus strain: structural evidence for a conserved genetic coupling with nitrile hydratase

et al. 1991

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A new enantiomer-selective amidase active on several 2-aryl propionamides was identified and purified from a newly isolated Rhodococcus strain. The characterized amidase is an apparent homodimer, each molecule of which has an Mr of 48,554; it has a specific activity of 16.5 g,mol of S(+)-2-phenylpropionic acid formed per min per mg of enzyme from the racemic amide under our conditions. An oligonucleotide probe was deduced from limited peptide information and was used to clone the corresponding gene, named amdA. As expected, significant homologies were found between the amino acid sequences of the enantiomer-selective amidase of Rhodococcus sp., the corresponding enzyme from Brevibacterium sp. strain R312, and several known amidases, thus confirming the existence of a structural class of amidase enzymes. Genes probably coding for the two subunits of a nitrite hydratase, albeit in an inverse order, were found 39 bp downstream of amdA, suggesting that such a genetic organization might be conserved in different microorganisms. Although we failed to express an active Rhodococcus amidase in Escherichia coli, even in conditions allowing the expression of an active R312 enzyme, the high-level expression of the active recombinant enzyme could be demonstrated in Brevibacterium lactofermentum by using a pSRl-derived shuttle vector.After a systematic search for soil microorganisms able to carry out the enantiomer-selective hydrolysis of racemic nitriles or amides, especially 2-aryl propionamides, we recently reported the purification and cloning of such an amidolytic activity from Brevibacterium sp. strain R312 (15). We found that the gene coding for the enantiomer-selective amidase, amdA, was closely linked to the genes coding for the two subunits, a and ,B, of nitrile hydratase, the enzyme responsible for the hydration of various nitriles to the corresponding amides (10). The structure of this region led to the hypothesis that the two genes could be translated from a polycistronic mRNA, thus supporting the view that there is a nitrile utilization operon, at least in this bacterium. These results have been confirmed by another group (9). Moreover, it was found that the identified amidase displays a striking homology to other known amidases, such as the acetamidase from Aspergillus nidulans and the indoleacetamide hydrolases from Pseudomonas syringae (savastanoi) and Agrobacterium tumefaciens. Interestingly, the homology was not scattered through the whole amino acid sequence but was mainly focused in a portion of the sequence around amino acids 150 to 220 of the R312 sequence (47 to 65% strict identity), strongly suggesting that this conserved sequence could be part of the active site of this class of enzymes.We now report the identification of a new Rhodococcus strain, obtained during a microbiological screening of soil samples, which is also characterized by efficient stereospecific amidase activity on 2-aryl propionamides. The enzyme responsible for this activity was purified, cloned, and sequenced. It is significantly ...

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Chemical synthesis of a gene coding for human angiogenin, its expression in Escherichia coli and conversion of the product into its active form

Cited by 56 publications

References 15 publications

Specific binding of angiogenin to calf pulmonary artery endothelial cells.

Specific binding of angiogenin to calf pulmonary artery endothelial cells.

Cis proline mutants of ribonuclease A. I. thermal stability

Purification, cloning, and primary structure of a new enantiomer-selective amidase from a Rhodococcus strain: structural evidence for a conserved genetic coupling with nitrile hydratase

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