High-level production of human α- and β-globins in insect cells

Groebe, Duncan R.; Busch, Mark R.; Tsao, Twee; Luh, Frederick Y.; Tam, Ming F.; Chung, Albert E.; Gaskell, Michael J.; Liebhaber, Stephen A.; Ho, Chien

doi:10.1016/s1046-5928(05)80097-x

Cited by 5 publications

(5 citation statements)

References 22 publications

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“…(Lower panel) mTOR-Flag protein levels were quantified by densitometry relatively to the β-actin levels, and these values were plotted for each condition (average and standard deviations [SDs] of three independent experiments) normalized to the HBB 5 ′ UTR-Flag levels in the control vehicle condition, arbitrarily set to 100%. the HBB 5 ′ UTR, may reflect the lower efficiency of the mTOR 5 ′ UTR in mediating cap-dependent translation when compared to the highly efficient human HBB 5 ′ UTR (Shakin and Liebhaber 1986;Groebe et al 1992). Together, these observations suggest that mTOR protein expression is resistant to perturbations of ternary and eIF4F complexes, and that the mTOR 5 ′ UTR is involved in this process.…”

Section: Resultsmentioning

confidence: 98%

Cap-independent translation ensures mTOR expression and function upon protein synthesis inhibition

Marques-Ramos

Candeias

Menezes

et al. 2017

RNA

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The mechanistic/mammalian target of rapamycin (mTOR) is a conserved serine/threonine kinase that integrates cellular signals from the nutrient and energy status to act, namely, on the protein synthesis machinery. While major advances have emerged regarding the regulators and effects of the mTOR signaling pathway, little is known about the regulation of gene expression. Here, we show that the human transcript can be translated in a cap-independent manner, and that its 5' untranslated region (UTR) is a highly folded RNA scaffold capable of binding directly to the 40S ribosomal subunit. We further demonstrate that is able to bypass the cap requirement for translation both in normal and hypoxic conditions. Moreover, our data reveal that the cap-independent translation of mTOR is necessary for its ability to induce cell-cycle progression into S phase. These results suggest a novel regulatory mechanism for gene expression that integrates the global protein synthesis changes induced by translational inhibitory conditions.

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Section: Resultsmentioning

confidence: 98%

Cap-independent translation ensures mTOR expression and function upon protein synthesis inhibition

Marques-Ramos

Candeias

Menezes

et al. 2017

RNA

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“…Human adult Hb (Hb A) is a tetrameric protein containing two a chains and two (3 chains having 141 and 146 amino acid residues each, respectively. Human globins and Hbs have been expressed in transgenic mice (1)(2)(3)(4), transgenic swine (5), insect cell cultures (6), yeast (7,8), and Escherichia coli (9)(10)(11). In many respects, the E. coli system is the best choice for our purposes because of its high expression efficiency and the ease ofperforming site-directed mutagenesis.…”

mentioning

confidence: 99%

Production of unmodified human adult hemoglobin in Escherichia coli.

Shen

Simplăceanu

et al. 1993

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

160

235

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We have constructed a plasmid (pHE2) in which the synthetic human a-and 3-globin genes and the methionine aminopeptidase (Met-AP) gene from Escherichia coli are coexpressed under the control of separate tac promoters. The Hbs were expressed in E. coli JM109 and purified by fast protein liquid chromatography, producing two major components, a and b. Electrospray mass spectrometry shows that at least 98% and about 90% of the expressed a and (3 chains of component a, respectively, have the expected masses.The remaining 10% of the P chain in component a corresponds in mass to the .8 chain plus methionine. In component b, both a and (3 chains have the correct masses without detectable N-terminal methionine (<2%). These results have been confirmed by Edman degradation studies of the amino-terminal sequences of the a and ,B chains of these two recombinant Hb (rHb) samples. rHbs from components a and b exhibit visible optical spectra identical to that of human normal adult Hb (Hb A). Component a and Hb A have very similar oxygen-binding properties, but component b shows somewhat altered oxygen binding, especially at low pH values. 1H-NMR spectra of component a and Hb A are essentially identical, whereas those of component b exhibit altered ring current-shifted and hyperfine-shifted proton resonances, indicating altered heme conformation in the (3 chain. These altered resonance patterns can be changed to those of Hb A by converting component b to the ferric state and then to the deoxy state and finally back to either the carbonmonoxy or oxy form. Thus, our E. coli expression system produces native, unmodified Hb A in high yield and can be used to produce desired mutant Hbs.To make use of our ability to rationally design mutant human Hbs needed for research on structure-function relationships, an efficient expression system for producing unmodified human Hbs in high yields is needed. Human adult Hb (Hb A) is a tetrameric protein containing two a chains and two (3 chains having 141 and 146 amino acid residues each, respectively. Human globins and Hbs have been expressed in transgenic mice (1-4), transgenic swine (5), insect cell cultures (6), yeast (7, 8), and Escherichia coli (9-11). In many respects, the E. coli system is the best choice for our purposes because of its high expression efficiency and the ease ofperforming site-directed mutagenesis. The first E. coli system to express human a-and 3-globin as a fusion protein was developed by Nagai and Th0gersen (9,12), but the product processing procedure is very laborious and gives low yield. Thus, this expression system has limitations, especially when large amounts of recombinant Hb (rHb) are required for biochemical-biophysical studies. Hoffman et al.

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“…These last two systems, however, result in globin chains containing N-terminal methionine, which may affect functional properties of hemoglobin. More recently, Shen et al (6) expressed soluble hemoglobin tetramers lacking N-terminal methionine in bacteria by coexpression of ␣-and ␤-globin cDNAs with methionine aminopeptidase cDNA.In order to further the understanding of hemoglobin assembly and folding, production of soluble single-chain hemoglobin variants is critical; however, expression of recombinant, soluble, individual globin chains has not been realized to date (4,5,7,8). Globins expressed in cells with and without additional hemin often form insoluble inclusion bodies that require harsh denaturing conditions for solubilization.…”

mentioning

confidence: 99%

“…In order to further the understanding of hemoglobin assembly and folding, production of soluble single-chain hemoglobin variants is critical; however, expression of recombinant, soluble, individual globin chains has not been realized to date (4,5,7,8). Globins expressed in cells with and without additional hemin often form insoluble inclusion bodies that require harsh denaturing conditions for solubilization.…”

mentioning

confidence: 99%

“…Globins expressed in cells with and without additional hemin often form insoluble inclusion bodies that require harsh denaturing conditions for solubilization. After solubilization, chains must then be renatured in vitro and form correctly folded native globin chains, which then must properly assemble to form authentic hemoglobin tetramers (1,(7)(8)(9). This process is labor intensive, and efficiency of tetramer reconstitution from denatured globin chains is very low.…”

mentioning

confidence: 99%

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Expression of Soluble Human β-Globin Chains in Bacteria and Assembly in Vitro with α-Globin Chains

Yamaguchi

Pang

Reddy

et al. 1996

Journal of Biological Chemistry

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Authentic soluble human ␤-globin chains were produced in Escherichia coli using an expression plasmid (pHE2␤) containing full-length cDNAs coding for human ␤-globin chain and methionine aminopeptidase. Spectral properties of the purified ␤-globin were identical to those of authentic ␤-globin. Soluble ␤-globin showed low (16 kDa) and high molecular mass (32 kDa) forms that could be separated by gel filtration chromatography. SDS-polyacrylamide gel electrophoresis and electrospray mass spectrometry revealed the 32-kDa species was dimeric ␤-globin formed by an intermolecular disulfide bond, while the 16-kDa species was authentic monomeric ␤-globin. Monomeric forms of ␤-globin, like authentic native ␤-globin, formed tetrameric hemoglobin (Hb) A (␣ 2 ␤ 2 ) in vitro upon incubation with ␣-globin, while dimeric forms did not. When ␤-globin dimers, however, were converted to monomers by incubation with dithiothreitol, the ␤-globin chain monomers assembled with ␣-globin and formed hemoglobin tetramers. ␣-Globin was more thermally unstable than ␤-globin, while assembled tetramers promoted higher stability. Disulfide-bonded ␤-globin dimers showed a slight increase in thermal stability compared with ␤-globin; however, dimers were still more unstable than tetrameric Hb A. These results indicate that presence of ␣ chains favors assembly with ␤-globin, ␤-␤ dimers cannot bind ␣ chains, and that Hb A tetramer formation results in the most thermally stable species.The development of molecular biological techniques to replace selectively individual amino acids has helped further our understanding of the relationship between structure and function of hemoglobin. Initial reports described production of normal and modified human ␤-globin in bacteria employing a fusion protein expression vector (1). An expression system was later developed in which ␣-and ␤-globin chains were coexpressed, resulting in formation of soluble tetrameric hemoglobin in yeast (2, 3). Recent studies described coexpression of human ␣-and ␤-globin in Escherichia coli, which resulted in formation of soluble tetrameric hemoglobins (4), as well as a system for high expression of insoluble ␤-globin chains in E. coli. (5). These last two systems, however, result in globin chains containing N-terminal methionine, which may affect functional properties of hemoglobin. More recently, Shen et al. (6) expressed soluble hemoglobin tetramers lacking N-terminal methionine in bacteria by coexpression of ␣-and ␤-globin cDNAs with methionine aminopeptidase cDNA.In order to further the understanding of hemoglobin assembly and folding, production of soluble single-chain hemoglobin variants is critical; however, expression of recombinant, soluble, individual globin chains has not been realized to date (4,5,7,8). Globins expressed in cells with and without additional hemin often form insoluble inclusion bodies that require harsh denaturing conditions for solubilization. After solubilization, chains must then be renatured in vitro and form correctly folded native globin chains, which...

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High-level production of human α- and β-globins in insect cells

Cited by 5 publications

References 22 publications

Cap-independent translation ensures mTOR expression and function upon protein synthesis inhibition

Cap-independent translation ensures mTOR expression and function upon protein synthesis inhibition

Production of unmodified human adult hemoglobin in Escherichia coli.

Expression of Soluble Human β-Globin Chains in Bacteria and Assembly in Vitro with α-Globin Chains

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