A method for construction of long randomized open reading frames and polypeptides

A method is presented for construction of randomized open readingframe sequences (ORFs) and gene libraries containing them. Thebuilding blocks for the ORFs were 75 bp long DNA fragments generatedby cloning sequences from a single synthetic oligonucleotidepreparation by bridge mutagenesis. The fragments had the propertythat, regardless of their orientation in the ligated product,the ORF of the construct was maintained. The heterogeneity ofthe ORFs resulted from the random ligation of 2000 differentDNA fragments. The randomized ORFs were cloned downstream fromthe lac promoter in a multicopy plasmid in Escherichia coli.To test the method, a library of 10⁶ clones was constructed.     

Modulation of enzyme activity by antibody binding to an alkaline phosphatase-epitope hybrid protein

An epitope from the HTV-1 gpl20 protein V3 loop has been insertedonto the surface of bacterial alkaline phosphatase at differentpositions in the vicinity of the enzyme active site, creatinghybrid proteins that can bind to an anti-gpl20 monoclonal antibody.One of the hybrid proteins, API1, has a 13 amino acid V3 loopsequence inserted between residues 407 and 408 of alkaline phosphatase.The enzymatic activity of this protein is modulated upon antibodybinding. API1 maintains the full activity of the wild type alkalinephosphatase but in the presence of the anti-gpl20 antibody,the enzyme activity is inhibited by 40–50%. Thus, thehybrid enzyme can be used to detect the presence of antibodyin solution. The concept of signalling proteins may have a wideapplication. Two models for the mechanism of modulation, sterichindrance and allosteric regulation, are discussed.     

Crystallization of (Fe, Co)78Si9B13 alloys: influence of relaxation processes

Crystallization of Fe78-xCoxSi9B13 amorphous alloys has been studied by differential scanning calorimetry (DSC), thermomagnetic gravimetry and X-ray diffraction techniques. Thermal stability of the amorphous alloy monotonically decreases with increasing cobalt content. The devitrification process as detected by DSC occurs in two main stages partially overlapping in temperature, but magnetic characterization reveals a third stage for cobalt-rich alloys. Primary precipitation of -(Fe, Co) is followed by a polymorphic reaction to give (Fe, Co)2B phase. Fe3B-type phase is also detected at the end of the first crystallization stage for iron-rich alloys and -Co(Si) and Co2B phases are found in fully crystallized samples for x=60. The influence of relaxation processes on the crystallization was investigated, but no significant effects of pre-annealing on the crystallization parameters resulted. © 1998 Chapman & Hall     

3-D structure of the D153G mutant of Escherichia coli alkaline phosphatase: an enzyme with weaker magnesium binding and increased catalytic activity

The substitution of aspartate at position 153 in Escherichiacoli alkaline phosphatase by glycine results in a mutant enzymewith 5-fold higher catalytic activity (k_cat but no change inKm at pH 8.0 in 50 mM Tris-HCl. The increased k_cat is achievedby a faster release of the phosphate product as a result ofthe lower phosphate affinity. The mutation also affects Mg²⁺binding, resulting in an enzyme with lower metal affinity. The3-D X-ray structure of the D153G mutant has been refined at2.5 Å to a crystallographic Rfactor of 16.2%. An analysisof this structure has revealed that the decreased phosphateaffinity is caused by an apparent increase in flexibility ofthe guanidinium side chain of Argl66 involved in phosphate binding.The mutation of Aspl53 to Gly also affects the position of thewater ligands of Mg²⁺, and the loop Glnl52–Thrl55 is shiftedby 0.3 Å away from the active site. The weaker Mg²⁺ bindingof the mutant compared with the wild type is caused by an alteredcoordination sphere in the proximity of the Mg²⁺ ion, and alsoby the loss of an electrostatic interaction (Mg²⁺.COO^-Aspl53)in the mutant Its ligands W454 and W455 and hydroxyl of Thrl55,involved in the octahedral coordination of the Mg²⁺ ion, arefurther apart in the mutant compared with the wild-type     

3-D structure of a mutant (Asp101 -> Ser) of E.coli alkaline phosphatase with higher catalytic activity

Mutagenesis of the absolutely conserved residue Asp101 of thenon-specific monoesterase alkaline phosphatase (E.C. 3.1.3.1 [EC] )from E.coli has produced an enzyme with increased k_cat. Thecarboxyl group of the Asp101 residue has been proposed to beinvolved in the positioning of Arg166 and the formation of thehelix that contains the active site Ser 102. The crystal structureof the Asp101     

Mutagenesis of conserved residues within the active site of Escherichia coli alkaline phosphatase yields enzymes with increased kcat

The likelihood for improvement in the catalytic properties ofEscherichia coli alkaline phosphatase was examined using site-directedmutagenesis. Mutants were constructed by introducing sequencechanges into nine preselected amino acid sites within 10 A ofthe catalytic residue serine 102. When highly conserved residuesin the family of alkaline phosphatases were mutated, many ofthe resulting enzymes not only maintained activity, but alsoexhibited greatly improved tra,. Of –170 mutant enzymesscreened, 5% (eight mutants) exhibited significant increasesin specific activity. In particular, a substitution by serineof a totally invariant AsplOl resulted in a 35-fold increaseof specific activity over wild-type at pH 10.0. Up to 6-foldincreases the k_cat/k_m ratio were observed.