TY  - JOUR
A1  - Gibbs, Moreland D.
A1  - Reeves, Rosalind A.
A1  - Sunna, Anwar
A1  - Bergquist, Peter L.
T1  - A yeast intron as a translational terminator in a plasmid shuttle vector
N2  - Plasmid shuttle vectors that contain both prokaryotic (Escherichia coli) and eukaryotic origins of replication are routinely used in molecular biology since E coli is generally the organism of choice for manipulation of recombinant DNA. Initial transformation of the shuttle vector into E coli allows production of microgram quantities of DNA suitable for transformation of low-transformationefficiency hosts. A shuttle/expression vector for the yeast Kluyveromyces lactis, pCWK1, allows recombinant protein fused to the killer toxin signal sequence to be secreted to the medium. The heterologous genes are transcribed under the control of the K lactis LAC4 promoter, which is tightly regulated in K lactis. However, in E coli the LAC4 promoter functions constitutively, and as a result, uncontrolled transcription and translation of genes that are toxic in E coli can result in cell death, and subsequent failure to recover intact E. coli transformants. We have constructed and tested a modified shuttle vector that contains a K lactis ribosomal intron that acts as a translational terminator in E coli, preventing or reducing the expression of recombinant proteins and avoiding toxicity. When transcribed in K lactis, the intron is spliced from the mRNA allowing the translation of intact full- length, active recombinant gene product. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved
Y1  - 2004
SN  - 1567-1356
ER  - 
TY  - JOUR
A1  - Roske, Y.
A1  - Sunna, A.
A1  - Pfeil, Wolfgang
A1  - Heinemann, Udo
T1  - High-resolution crystal structures of Caldiceflulosiruptor strain Rt8B.4 carbohydrate-binding module CBM27-1 and its complex with mannohexaose
N2  - Carbohydrate-binding modules (CBMs) are the most common non-catalytic modules associated with enzymes active in plant cell-wall hydrolysis. Despite the large number of putative CBMs being identified by amino acid sequence alignments, only few representatives have been experimentally shown to have a carbohydrate-binding function. Caldicellulosiruptor strain Rt8B.4 Man26 is a thermostable modular glycoside hydrolase beta-mannanase which contains two non-catalytic modules in tandem at its N terminus. These modules were recently shown to function primarily as beta- mannan-binding modules and have accordingly been classified as members of a novel family of CBMs, family 27. The N- terminal CBM27 (CsCBM27-1) of Man26 from Caldicellulosiruptor Rt8B.4 displays high-binding affinity towards mannohexaose with a K-a of 1 x 10(7) M-1. Accordingly, the high-resolution crystal structures of CsCBM27-1 native and its mannohexaose complex were solved at 1.55 Angstrom and 1.06 Angstrom resolution, respectively. In the crystal, CsCBM27-1 shows the typical beta-sandwich jellyroll fold observed in other CBMs with a single metal ion bound, which was identified as calcium. The crystal structures reveal that the overall fold of CsCBM27-1 remains virtually unchanged upon sugar binding and that binding is mediated by three solvent-exposed tryptophan residues and few direct hydrogen bonds. Based on binding affinity and thermal unfolding experiments this structural calcium is shown to play a role in the thermal stability of CsCBM27-1 at high temperatures. The higher binding affinity of CsCBM27-1 to mannooligosaccharides when compared to other members of CBM family 27 might be explained by the different orientation of the residues forming the "aromatic platform" and by differences in the length of loops. Finally, evidence is presented, on the basis of fold similarities and the retention of the position of conserved motifs and a calcium ion, for the consolidation of related CBM families into a superfamily of CBMs. (C) 2004 Elsevier Ltd. All rights reserved
Y1  - 2004
SN  - 0022-2836
ER  -