Improvement of the quality of lumazine synthase crystals by protein engineering.

Icosahedral macromolecules have a wide spectrum of potential nanotechnological applications, the success of which relies on the level of accuracy at which the molecular structure is known. Lumazine synthase from Bacillus subtilis forms a 150 A icosahedral capsid consisting of 60 subunits and crystallizes in space group P6(3)22 or C2. However, the quality of these crystals is poor and structural information is only available at 2.4 A resolution. As classical strategies for growing better diffracting crystals have so far failed, protein engineering has been employed in order to improve the overexpression and purification of the molecule as well as to obtain new crystal forms. Two cysteines were replaced to bypass misfolding problems and a charged surface residue was replaced to force different molecular packings. The mutant protein crystallizes in space group R3, with unit-cell parameters a = b = 313.02, c = 365.77 A, alpha = beta = 90.0, gamma = 120 degrees , and diffracts to 1.6 A resolution.

[The existence of contamination in enzymes used for the isolation of Aspergillus and Candida species DNA]. / Aspergillus ve Candida cinsi DNA'sinin izolasyonunda kullanilan enzimlerde kontaminasyon varliginin arastirilmasi.

Molecular biological methods which are widely used in different fields, have been replaced with conventional diagnostic tests for the early diagnosis of invasive fungal infections, recently. Polymerase chain reaction (PCR) is one of these methods, with high specificity and sensitivity, which is accepted throughout the world. However, the enzymes that are used for the isolation of target DNA, may be contaminated with the gene sequences of some other fungal species in the preparation steps and may affect the PCR results. The studies showed that, the contamination of these enzymes with any type of fungi during their production steps, leads false positive results in PCR tests. So, the additional studies are recommended for minimizing the contamination. The aim of this study was to search whether the enzymes necessary for the isolation of fungal target DNA, used in PCR, are contaminated or not. For this purpose, five different enzymes namely, Zymolase 20T (from Arthrobacter luteus, two examples from different companies), lyticase (from Arthrobacter luteus), lysing enzyme (from Trichoderma harzianum) and proteinase K (from Tritrachium album) have been investigated for the presence of contamination. As a result, both of zymolase 20T enzymes were found to be contaminated from some fungal species with the demonstration of 18S rRNA gene sequences, while the other enzymes were found non-contaminated. By the help of this method, the most suitable enzyme for PCR was chosen and fungal contamination was prevented.