Human DNA polymerase nu (Pol nu) is a conserved family A DNA polymerase of uncertain biological function. Physical and biochemical characterization aimed at understanding Pol nu function is hindered by the fact that, when over-expressed in Escherichia coli, Pol nu is largely insoluble, and the small amount of soluble protein is difficult to purify. Here we describe the use of high hydrostatic pressure to refold Pol nu from inclusion bodies, in soluble and active form. The refolded Pol nu has properties comparable to those of the small amount of Pol nu that was purified from the soluble fraction. The approach described here may be applicable to other DNA polymerases that are expressed as insoluble inclusion bodies in E. coli.
[Show abstract][Hide abstract] ABSTRACT: The fidelity of DNA synthesis by A-family DNA polymerases ranges from very accurate for bacterial, bacteriophage, and mitochondrial family members to very low for certain eukaryotic homologues. The latter include DNA polymerase ν (Pol ν) which, among all A-family polymerases, is uniquely prone to misincorporating dTTP opposite template G in a highly sequence-dependent manner. Here we present a kinetic analysis of this unusual error specificity, in four different sequence contexts and in comparison to Pol ν's more accurate A-family homologue, the Klenow fragment of Escherichia coli DNA polymerase I. The kinetic data strongly correlate with rates of stable misincorporation during gap-filling DNA synthesis. The lower fidelity of Pol ν compared to that of Klenow fragment can be attributed primarily to a much lower catalytic efficiency for correct dNTP incorporation, whereas both enzymes have similar kinetic parameters for G-dTTP misinsertion. The major contributor to sequence-dependent differences in Pol ν error rates is the reaction rate, k(pol). In the sequence context where fidelity is highest, k(pol) for correct G-dCTP incorporation by Pol ν is ~15-fold faster than k(pol) for G-dTTP misinsertion. However, in sequence contexts where the error rate is higher, k(pol) is the same for both correct and mismatched dNTPs, implying that the transition state does not provide additional discrimination against misinsertion. The results suggest that Pol ν may be fine-tuned to function when high enzyme activity is not a priority and may even be disadvantageous and that the relaxed active-site specificity toward the G-dTTP mispair may be associated with its cellular function(s).
[Show abstract][Hide abstract] ABSTRACT: Much knowledge has been gained for the last 30 years about the effects of pressure on bacteria, and various pressure-based technologies have been designed. The development of modern molecular biology techniques (e.g., DNA microarrays) as well as the technological advances realized in the manufacturing of robust sampling and high-pressure devices has allowed these advances. Not only the direct effects on cell components (membranes, proteins, and nucleic acids) have been unraveled, but also the cellular response to pressure has been investigated by means of transcriptome and proteome analyses. Initially, research was performed by marine biologists who studied the microorganisms living in the deep sea at pressures of 1,000 bar. In parallel, food technologists developed pressure-based methods for inactivating microorganisms without altering the food properties as much as with temperature treatment. The preservation of specific product properties is also the rationale for pressure-based methods for the disinfection of biomaterials and for vaccine production. Therefore, attention was first focused on the “killing” potential of high pressure. On the other hand, there has been a growing interest in using elevated pressures (up to ~10 bar) for enhancing the productivity of bioprocesses. In this case, no killing effect was sought, but pressure was applied to “boost” the process by enhancing the oxygen transfer to the cell culture. This paper gives an overview on the effects of pressures in the range of 1 bar to 10 kbar on bacteria and presents the major and most recent achievements realized in the development of pressure-based biotechnological applications.
[Show abstract][Hide abstract] ABSTRACT: Flavin reductase HpaC(St) catalyzes the reduction of free flavins using NADH or NADPH. High hydrostatic pressure was used for the solubilization and refolding of HpaC(St), which was expressed as inclusion bodies in Escherichia coli to achieve high yield in a flavin-free form. The refolded HpaC(St) was purified using Ni-affinity chromatography followed by a heat treatment, which gave a single band on SDS-PAGE. The purified refolded HpaC(St) did not contain FMN, unlike the same enzyme expressed as a soluble protein. After the addition of FMN to the protein solution, the refolded enzyme showed a higher activity than the enzyme expressed as the soluble protein. Crystals of the refolded enzyme were obtained by adding FMN, FAD, or riboflavin to the protein solution and without the addition of flavin compound.
Protein Expression and Purification 06/2012; 84(2):214-8. DOI:10.1016/j.pep.2012.06.006 · 1.70 Impact Factor
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