RESUMO
Using compartmentalized self-replication (CSR), we evolved a version of Pyrococcus furiosus (Pfu) DNA polymerase that tolerates modification of the γ-phosphate of an incoming nucleotide. A Q484R mutation in α-helix P of the fingers domain, coupled with an unintended translational termination-reinitiation (split) near the finger tip, dramatically improve incorporation of a bulky γ-phosphate-O-linker-dabcyl substituent. Whether synthesized by coupled translation from a bicistronic (-1 frameshift) clone, or reconstituted from separately expressed and purified fragments, split Pfu mutant behaves identically to wild-type DNA polymerase with respect to chromatographic behavior, steady-state kinetic parameters (for dCTP), and PCR performance. Although naturally-occurring splits have been identified previously in the finger tip region of T4 gp43 variants, this is the first time a split (in combination with a point mutation) has been shown to broaden substrate utilization. Moreover, this latest example of a split hyperthermophilic archaeal DNA polymerase further illustrates the modular nature of the Family B DNA polymerase structure.
Assuntos
Proteínas Arqueais/química , DNA Polimerase Dirigida por DNA/química , Sequência de Aminoácidos , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Evolução Molecular Direcionada , Dados de Sequência Molecular , Nucleotídeos/química , Nucleotídeos/metabolismo , Fosfatos/química , Engenharia de Proteínas , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
Escherichia coli maltose-binding protein (MBP) is frequently used as an affinity tag to facilitate the purification of recombinant proteins. An important additional attribute of MBP is its remarkable ability to enhance the solubility of its fusion partners. MBPs are present in a wide variety of microorganisms including both mesophilic and thermophilic bacteria and archaea. In the present study, we compared the ability of MBPs from six diverse microorganisms (E. coli, Pyrococcus furiosus, Thermococcus litoralis, Vibrio cholerae, Thermotoga maritima, and Yersinia pestis) to promote the solubility of eight different aggregation-prone proteins in E. coli. In contrast to glutathione S-transferase (GST), all of these MBPs proved to be effective solubility enhancers and some of them were even more potent solubilizing agents than E. coli MBP.
