Recombinant mouse prolactin: expression in Escherichia coli, purification and biological activity

1992 ◽  
Vol 8 (2) ◽  
pp. 165-172 ◽  
Author(s):  
M. Yamamoto ◽  
T. Harigaya ◽  
T. Ichikawa ◽  
K. Hoshino ◽  
K. Nakashima
Keyword(s):  
Escherichia Coli ◽  
Biological Activity ◽  
Amino Acid ◽  
Multicopy Plasmid ◽  
High Level Expression ◽  
E Coli ◽  
Expression In Escherichia Coli ◽  
Oligonucleotide Duplex ◽  
Efficient Expression ◽  
High Level

ABSTRACT Transformation of Escherichia coli cells with a recombinant plasmid containing modified mouse prolactin (mPRL) cDNA and a pKK223-3 vector resulted in efficient expression of mPRL protein. Cloned mPRL cDNA was modified by removing the 5′ non-translating sequence as well as the sequence which encoded the signal peptide of preprolactin for recombination. In addition, approximately 100 nucleotides of the 5′-terminal region of the cDNA, which include the ATG initiation codon and the following 31 codons of mature mPRL, were replaced by a chemically synthesized oligonucleotide duplex. The sequence of this duplex was chosen to be rich in AT without changing the amino acid sequence of the protein. The modified cDNA was finally inserted into the multicopy plasmid, pUC19, before high-level expression of mPRL in E. coli cells was obtained. Western blotting analysis of total protein from transformed E. coli cells showed that both 23 and 16kDa peptides were recognized by specific mPRL antisera. The purified and refolded 23 kDa protein exhibited a growth-stimulating effect on rat Nb 2 Node lymphoma cells, and was very similar to that of natural pituitary PRL.

scholarly journals Escherichia coli RcsA, a Positive Activator of Colanic Acid Capsular Polysaccharide Synthesis, Functions To Activate Its Own Expression

1999 ◽  
Vol 181 (2) ◽  
pp. 577-584 ◽  
Author(s):  
Wolfgang Ebel ◽  
Janine E. Trempy
Keyword(s):  
Escherichia Coli ◽  
Capsular Polysaccharide ◽  
Sequence Motif ◽  
Multicopy Plasmid ◽  
Transcriptional Fusion ◽  
High Level Expression ◽  
E Coli ◽  
Polysaccharide Synthesis ◽  
Positive Regulators ◽  
High Level

ABSTRACT Capsule (cps) gene expression in Escherichia coli is controlled by a complex network of regulators. Transcription of the cps operon is controlled by at least two positive regulators, RcsA and RcsB. We show here that RcsA functions to activate its own expression, as seen by the 100-fold-increased expression of arcsA::lacZ transcriptional fusion in strains with high levels of RcsA protein, either due to a mutation inlon or due to overexpression of RcsA from a multicopy plasmid. Expression of the rcsA::lacZfusion is increased by but not dependent on the presence of RcsB. In addition, the effects of H-NS and RcsB on the expression ofrcsA are independent of each other. A sequence motif, conserved between the E. coli cps promoter and theErwinia amylovora ams promoter and previously shown to be the RcsA-RcsB binding site, was identified in the rcsApromoter region and shown to be required for high-level expression ofrcsA.

scholarly journals High-level expression of human dihydropteridine reductase (EC 1.6.99.7), without N-terminal amino acid protection, in Escherichia coli

1989 ◽  
Vol 261 (1) ◽  
pp. 265-268 ◽  
Author(s):  
W L F Armarego ◽  
R G H Cotton ◽  
H H Dahl ◽  
N E Dixon
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Dihydropteridine Reductase ◽  
Terminal Amino Acid ◽  
High Level Expression ◽  
E Coli ◽  
Human Enzyme ◽  
Terminal Amino ◽  
High Level ◽  
Very High

The cDNA coding for human dihydropteridine reductase [Dahl, Hutchinson, McAdam, Wake, Morgan & Cotton (1987) Nucleic Acids Res. 15, 1921-1936] was inserted downstream of tandem bacteriophage lambda PR and PL promoters in Escherichia coli vector pCE30. Since pCE30 also expresses the lambda c1857ts gene, transcription may be controlled by variation of temperature. The recombinant plasmid in an E. coli K12 strain grown at 30 degrees C, then at 45 degrees C, directed the synthesis of dihydropteridine reductase to very high levels. The protein was soluble, at least as active as the authentic human enzyme, and lacked the N-terminal amino acid protection.

High-Fidelity Translation of Recombinant Human Hemoglobin in Escherichia coli

1998 ◽  
Vol 64 (5) ◽  
pp. 1589-1593 ◽  
Author(s):  
Michael J. Weickert ◽  
Izydor Apostol
Keyword(s):  
Escherichia Coli ◽  
Error Rates ◽  
High Fidelity ◽  
Human Hemoglobin ◽  
Heterologous Proteins ◽  
Expression Systems ◽  
High Level Expression ◽  
E Coli ◽  
Translation Error ◽  
High Level

ABSTRACT Coexpression of di-α-globin and β-globin in Escherichia coli in the presence of exogenous heme yielded high levels of soluble, functional recombinant human hemoglobin (rHb1.1). High-level expression of rHb1.1 provides a good model for measuring mistranslation in heterologous proteins. rHb1.1 does not contain isoleucine; therefore, any isoleucine present could be attributed to mistranslation, most likely mistranslation of one or more of the 200 codons that differ from an isoleucine codon by 1 bp. Sensitive amino acid analysis of highly purified rHb1.1 typically revealed ≤0.2 mol of isoleucine per mol of hemoglobin. This corresponds to a translation error rate of ≤0.001, which is not different from typical translation error rates found for E. coli proteins. Two different expression systems that resulted in accumulation of globin proteins to levels equivalent to ∼20% of the level of E. colisoluble proteins also resulted in equivalent translational fidelity.

High-level expression in Escherichia coli of calcium-binding domains of an embryonic sea urchin protein

Gene ◽  
1984 ◽  
Vol 31 (1-3) ◽  
pp. 155-164 ◽  
Author(s):  
Mark Muesing ◽  
Clifford D. Carpenter ◽  
William H. Klein ◽  
Barry Polisky
Keyword(s):  
Escherichia Coli ◽  
Sea Urchin ◽  
Calcium Binding ◽  
High Level Expression ◽  
Expression In Escherichia Coli ◽  
Binding Domains ◽  
High Level

scholarly journals Expression in Escherichia coli and characterization of a reconstituted recombinant 7Fe ferredoxin from Desulfovibrio africanus

1996 ◽  
Vol 314 (1) ◽  
pp. 63-71 ◽  
Author(s):  
Johanneke L. H. BUSCH ◽  
Jacques L. J. BRETON ◽  
Barry M. BARTLETT ◽  
Richard JAMES ◽  
E. Claude HATCHIKIAN ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Magnetic Circular Dichroism ◽  
Wild Type ◽  
E Coli ◽  
Excess Iron ◽  
Expression In Escherichia Coli ◽  
Type D

Desulfovibrio africanus ferredoxin III is a monomeric protein (molecular mass of 6585 Da) that contains one [3Fe-4S]1+/0 and one [4Fe-4S]2+/1+ cluster when isolated aerobically. The amino acid sequence consists of 61 amino acids, including seven cysteine residues that are all involved in co-ordination to the clusters. In order to isolate larger quantities of D. africanus ferredoxin III, we have overexpressed it in Escherichia coli by constructing a synthetic gene based on the amino acid sequence of the native protein. The recombinant ferredoxin was expressed in E. coli as an apoprotein. We have reconstituted the holoprotein by incubating the apoprotein with excess iron and sulphide in the presence of a reducing agent. The reconstituted recombinant ferredoxin appeared to have a lower stability than that of wild-type D. africanus ferredoxin III. We have shown by low-temperature magnetic circular dichroism and EPR spectroscopy that the recombinant ferredoxin contains a [3Fe-4S]1+/0 and a [4Fe-4S]2+/1+ cluster similar to those found in native D. africanus ferredoxin III. These results indicate that the two clusters have been correctly inserted into the recombinant ferredoxin.

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