Expression of toxic recombinant proteins for structural studies in the E. coli Lemo21(DE3) expression system

M. Trundová1, T. Kovaľ2, P. Kolenko2, K. Fejfarová2, J. Dohnálek1, 2

1Institute of Biotechnology AS CR, v.v.i., Vídeňská 1083, 142 20 Praha 4, Czech Republic

2Institute of Macromolecular Chemistry AS CR, v.v.i., Heyrovského nám. 2, 162 06 Praha 6, Czech Republic

Recombinant DNA technology as well as heterologous expression and purification systems available today would allow us to obtain any desired protein in a sufficient amount and in an appropriate quality for detailed structure characterization. Nevertheless, production of some proteins remains still problematic. One of the major obstacles is the toxic effect of some proteins on the host expression system. A special approach is required to solve this problem and there are different possible ways how to overcome these expression difficulties in various expression systems.       

The E. coli expression system is a well known and widely used system for heterologous protein production which has many advantages such as low cost, low time consumption, high yields of proteins, etc.  On the other hand, problems in expression of many types of proteins are frequent. The first step is to define or estimate the character of the protein toxicity. There are several aspects which would have to be taken into account: whether the codon composition is compatible with the production system, a possible detrimental function of soluble protein over-expressed in host system or high levels of insoluble protein production leading to a significant metabolic drain for cells.

In our study we used the E. coli Lemo21(DE3) system to achieve and subsequently optimize expression of a toxic recombinant protein from Legionella pneumophila. A gene for protein designated Lpn3 has been identified in the genome of the L. pneumophila as 3nucleotidase/nuclease and this protein is homologous to class I nucleases.

The gene for Lpn3 nuclease contains 882 bp and the whole protein consists of 285 amino acid residues with a molecular mass of 32 kDa. Sequence analysis shows that the first 27 amino acids of the N -terminal end of the protein represent a signal peptide, which directs the protein out of the bacterial cells and it is cleaved off during the transport.

Several vectors from the Oxford Protein Production Facility UK (OPPF-UK) were tested for optimal expression of Lpn3 nuclease in the E. coli Lemo21(DE3) expression system. The whole Lpn3 gene was cloned to the pOPINE vector, while the nucleotide sequence without the N-terminal signal peptide was inserted into pOPINE, pOPINMalE, pOPINDsbA, pOPINP, pOPINTolB, and pOPINS.

Successful Lpn3 expression in Lemo21 cells was achieved in the cases of pOPINMalE and pOPINP constructs, which are primarily determined for periplasmic expression of recombinant proteins. A key factor for optimization of expression conditions was determination of suitable concentration of rhamnose in the media. Optimal concentration causes complete suppression of basal expression before IPTG induction. Other important parameters were temperature and time of growth before and after inductions. All these steps were necessary to effectively tune the expression level of this difficult recombinant protein.

This publication is supported by the project „BIOCEV – Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University (CZ.1.05/1.1.00/02.0109), by the Ministry of Education, Youth and Sports of the Czech Republic (grant No. LG14009 and grant No. EE2.3.30.0029).