Thialysine utilization for protein synthesis by an exponentially growing E. coli culture

The extent of protein lysine substitution by thialysine in E. coli cells grown in media containing the analog depends on the time interval the cells are grown in the presence of analog and on the analog concentration in the medium. By calculating the percent of lysine substitution in newly synthesized proteins it was shown that this reaches, after one cell doubling in the presence of analog, a maximum which is 17% in the cells grown with 0.1 or 0.2 mM thialysine and 8% in cells grown with 0.05 mM thialysine. Proteins synthesized in the presence of analog in the concentration range 0.05-0.2 mM show similar stability to those synthesized in the absence of analog. The extent of analog incorporation into newly synthesized proteins, as regards both the time course and the dependence on analog concentration in the medium, is strictly related to the extent of the repression of AK III, the first enzyme of lysine biosynthetic pathway.     

Thialysine and selenalysine utilization for protein synthesis by E. coli in comparison with lysine

Utilization of thialysine and selenalysine for protein synthesis by a lysine requiring E. coli mutant was studied. Incorporation into proteins of thialysine or selenalysine, added to culture medium together with lysine, becomes evident when the amount of available lysine in the medium is highly reduced, that is the mutant utilizes the isologs only after all the available natural aminoacid has been utilized. Compared to selenalysine, thialysine is better utilized; when both isologs are present in the medium at equal concentrations, up to 46% of protein lysine is substituted by thialysine and only 12% by selenalysine.     

Thialysine and selenalysine incorporation into CHO cell proteins and its effects on cell growth

CHO cells can incorporate into proteins both thialysine and selenalysine when both are present together in the culture medium. Thialysine and selenalysine inhibit cell growth and cell viability. The inhibitory effect of either analog is additive. The inhibition of cell viability is related to the extent of protein lysine substitution by thialysine or selenalysine; it is however irrelevant whether lysine is substituted by one or the other analog or by both.     

Thialysine utilization by a lysine-requiring Escherichia coli mutant

Summary Thialysine cannot completely substitute lysine as growth factor for a lysine-requiring E. coli mutant. However it can be utilized for growth in the presence of limiting amounts of lysine, in substitution of, and in competition with this latter. The effects of thialysine on growth rate, protein synthesis rate and cell viability, and its incorporation into proteins were studied in function of lysine and thialysine concentration in the culture media. Up to 60% of protein lysine substitution by thialysine is observed, without appreciable effects on cell viability.     

Thialysine utilization by thialysine resistant CHO cells

Thialysine resistant CHO cells utilize thialysine added to the culture medium to a lesser extent than the parental cells. Thialysine is utilized in protein synthesis and it is incorporated into proteins in place of lysine. The parental strain substitutes up to 11% of protein lysine by thialysine, while variant cells substitute a maximum of 5% of protein lysine.     

Thialysine utilization for protein synthesis by CHO cells

Chinese Hamster Ovary (CHO) cells utilize thialysine when added to the culture medium. Thialysine utilization is prevented by increasing lysine concentration in the medium, thus indicating that thialysine is utilized in substitution for and in competition with lysine. Almost all thialysine disappeared from the medium is recovered in cell protein hydrolysates. Thialysine is used for protein synthesis in substitution for lysine, and up to 10% of lysine can be substituted.     

Selenalysine utilization for growth and protein synthesis by a lysine requiring E. coli mutant

Summary Selenalysine can be utilized in substitution of lysine by a lysine requiring E. coli mutant. The presence of some lysine in the culture medium is necessary to allow selenalysine utilization for growth; in the presence of an excess of lysine, selenalysine is not utilized. When utilized, selenalysine gives rise to an increase of final growth. However, it shows some toxic effects as demonstrated by the decrease of both growth rate and cell viability. Selenalysine is incorporated into proteins in substitution of lysine. Up to a maximum of 50% of total protein lysine can be substituted. The decrease of cell viability is correlated with the extent of lysine substitution.This paper is dedicated to Professor A. E. Braunstein on his 80th birthday.     

重组人钙调素的制备及对细胞增殖作用影响的研究

The gene coding for human CaM was amplified by PCR in which pUC/hCaM3 cDNA was usd as template. After inserting the hCaM III cDNA into the expression plasmid pBV220, we constructed the hCaM3 cDNA-recombinant expression vector(hCaM3/pBV220). The recombinant plasmid was then transformed into E. coli DH5 alpha. After heat induction, a high level expression of CaM protein was obtained. SDS-PAGE analysis showed that the recombinant E. coli could express a 17 kD protein which accounted for about 20% of the total cellular protein. Western blot analysis showed that anti-CaM monoclonal antibody(McAb) specifically bound to the 17 kD band of expression product. rhCaM was purified by Phenyl-sepharose CL-4B affinity chromatography from recombinant bacterial lysate. 3-4 mg of the purified protein were obtained from 1 liter of bacterial culture. The rhCaM was able to activate NAD kinase to the same extent as the standard human brain CaM (Sigma). K562 cells and SP2/0 cells were seeded in 24-well or 96-well plate and cultured for 48 h with rhCaM and CaM-antagonist trifluoperazine(TFP). Cell proliferation rates was determined by MTT assay. There was a significant positive correlation between the concentrations of rhCaM and the cell proliferation rates. CaM-antagonist TFP had an inhibitory effect on cell proliferation rate. The inhibition could be corrected by the addition of extracellular rhCaM.     

Glucose metabolism at high density growth of E. coli B and E. coli K: Differences in metabolic pathways are responsible for efficient glucose utilization in E. coli B as determined by microarrays and northern blot analyses

The original article to which this Erratum refers was published in Biotechnol Bioeng 2005;90:805–820     

Positioning of chemosensory clusters in E. coli and its relation to cell division

Chemotaxis receptors and associated signalling proteins in Escherichia coli form clusters that consist of thousands of molecules and are the largest native protein complexes described to date in bacteria. Clusters are located at the cell poles and laterally along the cell body, and play an important role in signal transduction. Much work has been done to study the structure and function of receptor clusters, but the significance of their positioning and the underlying mechanisms are not understood. Here, we used fluorescence imaging to study cluster distribution and follow cluster dynamics during cell growth. Our data show that lateral clusters localise to specific periodic positions along the cell body, which mark future division sites and are involved in the localisation of the replication machinery. The chemoreceptor cluster positioning is thus intricately related to the overall structure and division of an E. coli cell.     

Immobilization of E. coli beta-galactosidase and its derivatives by polyacrylamide gel

We have recently prepared some crosslinked derivatives of Escherichia coli beta-galactosidase by treating the enzyme with bisimidoesters. In this article, we report the results obtained when the native and these crosslinked derivatives are entrapped in polyacrylamide gel lattice. It was found that use of combination of three protective agents, viz., bovine serum albumin, cysteine, and lactose, during immobilization gave an increased yield of 190% in the case of DMA crosslinked preparation. In the case of native enzyme, the K(m), pH optimum, and temperature optimum were found to remain unchanged on immobilization. The DMA crosslinked preparation entrapped in polyacrylamide in the presence of BSA, lactose, and cysteine was found to be a significantly better catalyst and hydrolyzed 47% milk lactose as compared to 31% hydrolysis by entrapped native enzyme in 6 h.     

Protective effects of sodium selenite on killing and mutation by N-methyl-N'-nitro-N-nitrosoguanidine in E. coli.

Sodium selenite was found to protect Escherichia coli cells against killing and mutagenic effects of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Such protective effects were not observed when cells were treated with N-methyl-N-nitrosourea (MNU). The protection by sodium selenite was not controlled by the ada gene, which is responsible for the repair of alkylated damage in DNA. A reduction of the amount of glutathione was found when cells were treated with sodium selenite, and glutathione is known to be involved in the methylation of DNA by MNNG, not by MNU. Reduced methylation by MNNG due to the reduction of the amount of glutathione caused by abundant sodium selenite was suggested to be the mechanism of protection.     

Prediction of effects of amino acid supplementation on growth of E. coli B/r

A mathematical model for the growth of a single cell of E. coli on medium containing amino acid is presented. A mixture of purified amino acids (glutamate, aspartate, serine, tyrosine, and leucine) combined in the ratios found in a natural digest (casein) were employed as the nitrogen source. Each of these amino acids is the representative of a different family of amino acids. The transport mechanisms and assimilation routes for each amino acid were inserted into the prototype model. The enzyme activities and saturation constants used in the model were based on literature data. The maximum velocities for uptake systems were calculated from experimental data. The formation and homeostasis of amino acid pools were regulated through cross-control of the activities of biosynthetic enzymes and of membrane transport of exogenous nutrients. The size of each amino acid pool was determined with mass balance equations that included terms for a transport system, a biosynthesis system, a transaminase enzyme system for interchange between the amino acid families, and a consumption system. The predictions of the extended model with regard to nutrient concentrations and growth rates compared well with the experimental data.