Title:Development of an Autoinducible Plasmid for Recombinant Protein
Production
Volume: 28
Issue: 12
Author(s): Young Kee Chae*Hakbeom Kim
Affiliation:
- Department of Chemistry, Sejong University, 209 Neungdong-Ro, Gwangjin-Gu, Seoul 05006, Korea
Keywords:
Autoinducible, recombinant protein, stationary phase, promoter, IPTG, protein production.
Abstract: Background: The production of recombinant proteins in E. coli involves such factors as
host strains, expression vectors, culture media, and induction methods. The typical procedure to
produce heterologous proteins consists of the following: (1) insertion of the target gene into a suitable
vector to construct an overexpression plasmid, (2) transformation of a strain specialized for
protein production with the constructed plasmid DNA, (3) growth of the host in a suitable medium
and induction of the protein production at a right moment, and (4) further growth to get the maximum
yield. There are hurdles involved in each of these steps, and researchers have developed
many materials or methods, which often require special recipes or procedures.
Objective: To eliminate the special requirements for recombinant protein production by using readily
available materials. Also to save time and effort in the routine protein production work.
Methods: We started with a vector capable of producing a target protein fused to the C-terminus of
the maltose-binding protein (MBP). The mCherry (red fluorescent protein) gene was fused to
MBP. It acted as a reporter in the initial screening procedure. The original lethal gene (barnase)
was replaced with sacB. We chose 3 stationary phase promoters and made hybrids of them by mixing
halves from each one. The T5 promoter was replaced with these stationary phase promoters or
their hybrids. The best plasmid was selected by the color intensity of the cell pellet. MBP and GST
genes were inserted in the place of sacB, and their production yields were compared with the original
plasmid in the conventional way of expression.
Results: We constructed an expression plasmid with an autoinducible promoter working in a host
that was not specially designed for protein production and in a TB medium that did not contain any
secret ingredient, nor was it difficult to prepare unlike Studier’s defined medium. This plasmid also
contains a color indicator that turns red when protein production is successful. We tested our system
with the maltose-binding protein (MBP) and the glutathione S-transferase (GST), and showed
that both proteins were produced to a level comparable to what the commercial medium and/or the
specialized strain yielded.
Conclusion: We developed a plasmid equipped with an autoinducible promoter, a hybrid of the
two promoters which were activated at the stationary phase. This plasmid does not need a special
E. coli strain nor a sophisticated nor an expensive medium. It produces an intense red (or pink) color,
which can be used as an indicator of a successful production of the target protein and as a predictive
measure of the amount of the produced target protein. We speculate that this plasmid will
have its greatest advantage when growing cells at low temperatures, which would inevitably take a
long time.
