Pfu DNA polymerase is a vital enzyme in PCR-related experiments. However, it is not easy to achieve high-level expression and high purity through one-step purification. This paper illustrates the method to acquire the full-length open reading frame of Pfu DNA polymerase. Without altering its amino acids, we have modified the codon usage, based on that of the enhanced green fluorescence protein (eGFP), and named it rPfu. The synthesized rPfu gene has been subcloned into the pET28a plasmid and expressed in four Escherichia coli strains without the pLysS plasmid. Three strains have expressed a high level of soluble Pfu DNA polymerase. With the aid of Ni-NTA Hisâ¢Bind® resin, we could obtain high purity (>95%) soluble recombinant protein. Compared with the commercial, proofreading DNA polymerase, rPfu's bioactivity was 12,987 U/mg; that is, 88,311 U of rPfu could be obtained from 50 mL cultured E. coli. The purified rPfu was able to amplify the length of DNA fragments at least 5.5 kb. The method of increasing soluble protein's yield using the eGFP codon usage may introduce a new possibility to the expression of other soluble recombinant proteins.
Codon Usage , Escherichia coli , Escherichia coli/genetics , Escherichia coli/metabolism , Recombinant Proteins , DNA-Directed DNA Polymerase/chemistry , DNA-Directed DNA Polymerase/genetics , DNA-Directed DNA Polymerase/metabolism
The crystal structure of LiMg(H(2)O)(2)[BP(2)O(8)]·H(2)O consists of tubular structural units, built from tetra-hedral (∞) (1){[BP(2)O(8)](3-)} borophosphate ribbons and (LiO(4))(n) helices running along [001], which are inter-connected by MgO(4)(H(2)O)(2) octa-hedra, forming a three-dimensional network structure with one-dimensional channels along [001] in which the water mol-ecules are located. The water mol-ecule in the channel is significantly displaced by up to 0.3â Å from the special position 6b (..2) to a half-occupied general position. Mg, B and one Li atom all lie on twofold axes. Of the two Li positions, one is at a special position 6b (..2), while the other is at a general position; both are only half-occupied.
