Glicyne Betaine transmethylase gene of Pseudomonas aeruginosa sequence analysis to overexpress the protein

FORRELLAD, MA; MASSIMELLI, MJ; BEASSONI, PR; BARRA, JL; GARRIDO, MN; LISA, AT

Iguazú, Misiones

Congreso; XL Reunión Anual, Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2004

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB)

Glycine betaine transmethylase gene (gbt) had been identified in P. aeruginosa Fildes III. Based on DNA sequence analysis, it was located in the published genome of PAO1 as PA3082 gene. Analysis of this locus revealed two putatives overlapping ORFs in different frames, ORF1 (1.965 bp) and ORF3 (1.542 bp) with an ATG methionine and a CTG leucine initiation codon, respectively.  The aim of our work is to characterize the GBT protein after its overexpression in an adequate vector. In the present work, we study which ORF encodes this protein. Different sequences, named M, T and J, in frame with ORF3 were amplified by PCR using Taq polymerase and subcloned in pET-15b expression vector. Efforts to overexpress the His-Tag fusion proteins in E. coli BL21(DE3) were unsuccessful. This failed protein expression was explained after DNA sequencing since a stop codon was observed as a consequence of transition mutation in ORFM and T.  ORFJ presented a deletion in two nucleotides. High fidelity Pfu Turbo polymerase was used to amplified ORFT and ORF1, in order to reduce possible mistakes introduced by Taq polymerase. After sequencing, the same transition mutation was observed in both ORFs. These results suggested that Taq did not introduce the mutation, but it was present in P. aeruginosa Fildes III gene. Then, ORF1 would encode and allow the overexpression of a functionally GBT protein in spite of the transition mutation