Constructing Prokaryotic ubiquitin protein expression vector with dual-tag by Gibson Assembly system

Abstract

Objective Constructing Pup (prokaryotic ubiquitin-like protein) expression vector is key point for study pupylation in vitro.In order to improve effective and specificity of protein purification, we need to construct Pup expression with his6 and strep tag, which will establish basis for further study on regulation of Pup-proteasome system under stress in mycobacteria. Methods Designing and synthesizing his6-strep fragments, amplifying and enriching them.The Pup fragment was amplified from Mycobacterium tuberculosis H37Rv genome.His6-strep and Pup genes were inserted into pET28a and shuttle plasmid pMV261 based on homologous recombination by Gibson Assembly system, constructing dual-tags vector, named as pET28a-his6-strep-Pup and pMV261-his6-strep-Pup.The right vector checked by polymerase chain reaction (PCR) and sequencing was transformed into E. coli and M. sm.These strains containing right vector were cultured and enriched, separately collecting pellet and supernatant after lyse by ultra-sonic.After sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) electrophoresis, the expression of dual-tags were checked by western blot. Results The vectors expressing dual-tags Pup were constructed successfully after clarified by PCR and sequencing analysis.The outcome of Western blot indicated positive expression of dual-tags protein separately in E. coli and M. sm and many Pupylation protein were found in M. sm. Conclusions The dual-tag vectors expressing his6 and strep are constructed rapidly and efficiently.These vectors can be used to purifying Pupylation protein and study their functions, which will establish the good base for the further investigating regulation of Pup-proteasome system under the stress in mycobacteria. Key words: Blotting, Western; Gibson Assembly; Homologous recombination; Tag protein; Pup