Targeting of interleukin-2 to the periplasm of Escherichia coli

Abstract

A synthetic gene coding for interleukin-2 (IL-2) was used to produce large amounts of recombinant IL-2 (met-IL-2) in Escherichia coli. Met-IL-2 was found to accumulate in the cytoplasm in an insoluble, aggregated form. Inclusion bodies located at the pole caps of cells were detected using immunogold labelling. Constructs were designed to fuse the IL-2 gene to DNA fragments encoding signal peptides for an outer-membrane protein (OmpA) or for a periplasmic protein (PhoA) of E. coli. No significant maturation was observed with these fusion proteins which were found in an insoluble form in the cytoplasm. The influence of charge disposition at the N-terminus of the mature portion of the protein was investigated by replacing positively charged amino acids with glutamic acid. None of the introduced substitutions had any effect. Various factors that might affect expression, secretion and folding were examined in an attempt to obtain secretion. By fusing IL-2 to the precursor maltose-binding protein (preMBP) a large fraction of the preMBP-IL-2 protein was correctly processed and transported to the periplasmic space. IL-2 derived from MBP-IL-2 after FXa cleavage possessed similar specific activity to recombinant IL-2 produced in Chinese Hamster ovary cells.

Directed evolution of super-secreted variants from phage-displayed human Interleukin-2

“…Neither the introduction of N - and C -terminal fusions and artificial disulphide bonds nor the mutational disruption of the negatively charged cavity at tail produced the expected results, leading to the view that IL-2 aggregation propensity is too high to be conquered through engineering 23 . Changing the charge near the cleavage site between the signal peptide and the N -terminus of IL-2 (replacing K8/K9 by acidic residues) did not result in periplasmic secretion in E. coli host 42 . Our results illustrate the advantages of blind directed evolution to manipulate complex molecular interaction networks where the effects of individual modifications cannot be easily predicted.…”

Lekteplase — a Secreted Tissue Plasminogen Activator Derivative from Escherichia coli

“…Some fusion protein can be exported to the periplasm, interleukin-2, when preMalK signal peptide was used. For the same protein, accumulation in the cytoplasm was observed in case of signal OmpA or PhoA [22]. Hence the amino acids sequence of mature protein may be involved in directing of mature molecule to the environment.…”