LB Crystallization and Preliminary X-ray Diffraction Analysis of L-Asparaginase from Rhodospirillum rubrum

Abstract

Published by United Scientific Group AbstractProtein X-ray crystallography will remain the most powerful method to obtain the protein 3D atomic structures in foreseeable future. However, the production of the protein crystal as well as it quality (order, intensity of diffraction, radiation stability) remains the major problem. Many important proteins including those of life science interest and pharmaceutical industry impact are difficult to crystallize. The second major problem in protein crystallography is radiation damage of obtaining crystals which can only be partially overcome by existing methods. In the present work we use the protein LB nanotemplate crystallization methodgeneralized procedure for triggering of crystallization of any given protein, which allows to obtain radiation stable and high quality diffracting crystals for further X-ray analysis by synchrotron radiation. We apply LB nanotemplate method to crystallization of L-asparaginase from Rhodospirillum rubrum. This protein has potential application for combined chemical and enzymatic therapy of malignant blood disorders and therefore for new anticancer drug development. We also compare the diffraction quality of asparagines crystal obtained by classical method and LB nanotemplate and report preliminary X-ray diffraction characterization by synchrotron radiation.

A Novel L-Asparaginase from Hyperthermophilic Archaeon Thermococcus sibiricus: Heterologous Expression and Characterization for Biotechnology Application

“…AAP92666.3) [ 55 , 56 ] and Rhodospirillum rubrum (GenBank accession No. QXG80441.1) [ 57 , 58 , 59 ] is about 27% and 33%, respectively. The identity between TsA and L-ASNases, FDA-approved for use as antileukemic agents, from E. coli (UniProtKB accession number P00805, marketed under the brand name Elspar, GenBank accession No.…”

“…and shares a low level of sequence identity with mesophilic L-ASNases. Available crystallographic data of L-ASNases of various origins indicate that, despite the low degree of homology, which may not exceed 20–30%, they have conserved regions that determine the similarity of their tertiary structures and the catalytic mechanism [ 49 , 54 , 59 , 64 ]. According to the amino acid sequence alignments, TsA displayed eight conserved residues ( Figure 1 b) essential for the catalytic activity, namely Thr12, Tyr22, Ser55, Thr56, Thr86, Asp87, Lys157 and Glu279.…”

Highly Active Thermophilic L-Asparaginase from Melioribacter roseus Represents a Novel Large Group of Type II Bacterial L-Asparaginases from Chlorobi-Ignavibacteriae-Bacteroidetes Clade

“…AAP92666.3) [32,33] and Rhodospirillum rubrum (GenBank accession No. QXG80441.1) [34][35][36] is about 32.7% and 41.7%, respectively. The identity between MrAII and FDA-approved L-ASNases from E. coli (UniProtKB accession number P00805, marketed under the brand name Elspar, GenBank accession No.…”