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Novel in-frame two codon translational hop during synthesis of bovine placental lactogen in a recombinant strain of Escherichia coli.

ABSTRACT: A recombinant Escherichia coli strain was constructed for the overexpression of bovine placental lactogen (bPL), using a bPL structural gene containing 9 of the rare arginine codons AGA and AGG. When high level bPL synthesis was induced in this strain, cell growth was inhibited and bPL accumulated to less than 10% of total cell protein. In addition, about 2% of the recombinant bPL produced from this strain exhibited an altered trypsin digestion pattern. Amino acid residues 74 through 109 normally produce 2 tryptic peptides, but the altered form of bPL lacked these two peptides and instead had a new peptide which was missing arginine residue 86 and one of the two flanking leucine residues. The codon for arginine residue 86 was AGG and the codons for the flanking leucine residues 85 and 87 were TTG. When 5 of the 9 AGA and AGG codons in the bPL structural gene were changed to more preferred arginine codons, cell growth was not inhibited and bPL accumulated to about 30% of total cell protein. When bPL was purified from this modified strain, which included changing the arginine codon at position 86 from AGG to CGT, none of the altered form of bPL was produced. These observations are consistent with a model in which translational pausing occurs at the arginine residue 86 AGG codon because the corresponding arginyl-tRNA species is reduced by the high level of bPL synthesis, and a translational hop occurs from the leucine residue 85 TTG codon to the leucine residue 87 TTG codon. This observation represents the first report of an error in protein synthesis due to an in-frame translational hop within an open reading frame.

SUBMITTER: Kane JF

PROVIDER: S-EPMC334590 | biostudies-other | 1992 Dec

REPOSITORIES: biostudies-other

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Novel in-frame two codon translational hop during synthesis of bovine placental lactogen in a recombinant strain of Escherichia coli.

Kane J F JF Violand B N BN Curran D F DF Staten N R NR Duffin K L KL Bogosian G G

Nucleic acids research 19921201 24

A recombinant Escherichia coli strain was constructed for the overexpression of bovine placental lactogen (bPL), using a bPL structural gene containing 9 of the rare arginine codons AGA and AGG. When high level bPL synthesis was induced in this strain, cell growth was inhibited and bPL accumulated to less than 10% of total cell protein. In addition, about 2% of the recombinant bPL produced from this strain exhibited an altered trypsin digestion pattern. Amino acid residues 74 through 109 normall ...[more]

PMID: 1480491

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Project description:BackgroundThe expression of heterologous proteins in Escherichia coli is strongly affected by codon bias. This phenomenon occurs when the codon usage of the mRNA coding for the foreign protein differs from that of the bacterium. The ribosome pauses upon encountering a rare codon and may detach from the mRNA, thereby the yield of protein expression is reduced. Several bacterial strains have been engineered to overcome this effect. However, the increased rate of translation may lead to protein misfolding and insolubilization. In order to prove this assumption, the solubility of several recombinant proteins from plants was studied in a codon bias-adjusted E. coli strain.ResultsThe expression of eight plant proteins in Escherichia coli BL21(DE3)-pLysS and BL21(DE3)-CodonPlus-pRIL was systematically studied. The CodonPlus strain contains extra copies of the argU, ileY, and leuW tRNA genes, which encode tRNAs that recognize the codons AGA/AGG, AUA and CUA, respectively (RIL codons). The level of expression and solubility of the recombinant proteins were analyzed by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting. We found that for all proteins the solubility was at least 25% in the BL21(DE3)-pLysS strain. However, when expressed in the BL21(DE3)-CodonPlus-pRIL strain, proteins having more than 5% of amino acids coded by RIL codons were localized mainly in the insoluble fraction. Also, their expression caused retarded growth and low cell yield in the codon bias-adjusted strain at all temperatures tested. On the contrary, the solubility of proteins containing less than 5% of amino acids coded by RIL codons remained unchanged in both strains and their expression caused no effect on cell growth.ConclusionOur results show that the expression of heterologous proteins coded by high RIL codon content coding sequences in a codon bias-adjusted strain is detrimental for their solubility. Our data support the hypothesis that the possible elimination of translational pauses that increase translation rate leads to protein misfolding and aggregation. This stresses the importance of strain selection according to codon content in any scheme where a large amount of biologically active product is desirable.

Project description:BackgroundBovine trophoblast binucleate cells (BNC) express a plethora of molecules including bovine placental lactogen (bPL, gene name is bCSH1) and bovine prolactin-related protein-1 (bPRP1). BCSH1 and bPRP1 are members of the growth hormone (GH)/prolactin (PRL) gene family, which are expressed simultaneously in BNC and are central to placentation and the progression of pregnancy in cattle. However, there is a paucity of information on the transcriptional regulatory mechanisms of both the bCSH1 and bPRP1 genes. Recent studies, however, have demonstrated that the expression of a number of genes is controlled by the methylation status of their promoter region. In the present study, we examined the cell-type-specific epigenetic alterations of the 5'-flanking region of the bCSH1 and bPRP1 genes to gain an insight into their regulatory mechanisms.ResultsAnalysis of 5-aza-2'-deoxycytidine treatment demonstrated that bCSH1 expression is moderately induced in fibroblast cultures but enhanced in BT-1 cells. Sodium bisulfite based sequencing revealed that bCSH1 is hypomethylated in the cotyledonary tissue but not in the fetal skin, and this pattern was not altered with the progression of pregnancy. On the other hand, the methylation status of bPRP1 was similar between the cotyledon and fetal skin. The bPRP1 gene was exclusively hypermethylated in a bovine trophoblast cell-derived BT-1 cell-line. While the activity of bCSH1 was similar in both BT-1 and bovine fibroblast cells, that of bPRP1 was specific to BT-1. Treatment with a demethylating agent and luciferase assays provided in vitro evidence of the positive regulation of bCSH1 but not bPRP1.ConclusionThis is the first report to identify the differential regulatory mechanisms of the bCSH1 and bPRP1 genes and indicates that bCSH1 might potentially be the only transcript that is subject to DNA methyltransferase regulation. The data indicates the possibility of novel kinetics of induction of the synchronously expressed BNC-specific bCSH1 and bPRP1 transcripts, which may aid the understanding of the intricate regulation and specific role(s) of these important molecules in bovine placentogenesis and the progression of pregnancy.

Project description:Placental lactogen (hPL) is a key hormone of pregnancy responsible for inducing maternal adaptations critical for a successful pregnancy. Low levels of placental lactogen have been associated with lower birth weight as well as symptoms of maternal depression and anxiety. Lower placental lactogen has been reported in women with higher body mass index (BMI) but it is unclear whether prenatal health behaviours predict hPL levels or if hPL is associated with infant weight outcomes. This study utilised data from the longitudinal Grown in Wales cohort, based in South Wales. Participants were recruited at the pre-surgical appointment for an elective caesarean section. This study incorporates data from recruitment, post-delivery and a 12 month follow-up. Measures of maternal serum hPL were available for 248 participants. Analysis included unadjusted and adjusted linear and binary regression. Unadjusted, prenatal smoking and a Health Conscious dietary pattern were associated with hPL levels, however this was lost on adjustment for BMI at booking, Welsh Index of Multiple Deprivation (WIMD) score and placental weight. When stratified by maternal BMI at booking, a Health Conscious dietary pattern remained associated with increased hPL levels in women with a healthy BMI (p=.024, B=.59. 95% CI=.08,1.11) following adjustment for WIMD score and placental weight. When adjusted for a wide range of confounders, maternal hPL was also associated with increased custom birthweight centiles (CBWC) (p=.014, B=1.64. 95% CI=.33,2.94) and increased odds of large for gestational age deliveries (p=<.001, Exp(B)=1.42. 95% CI=1.17,1.72). This study identified that consuming a Health Conscious dietary pattern in pregnancy was associated with increased hPL, within women of a healthy BMI. Moreover, higher hPL levels were associated with increased CBWC and increased odds of delivering a large for gestational age infant. This improves the current limited evidence surrounding the nature of hPL in these areas.

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Novel in-frame two codon translational hop during synthesis of bovine placental lactogen in a recombinant strain of Escherichia coli.

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Novel in-frame two codon translational hop during synthesis of bovine placental lactogen in a recombinant strain of Escherichia coli.

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