Strategies for the production of difficult-to-express full-length eukaryotic proteins using microbial cell factories: production of human alpha-galactosidase A

Abstract

22Obtaining high levels of pure proteins remains the main bottleneck of many scientific and 23 biotechnological studies. Among all the available recombinant expression systems Escherichia coli facilitates 24 gene expression by its relative simplicity, inexpensive and fast cultivation, well-known genetics and the large 25 number of tools available for its biotechnological application. However, recombinant expression in E. coli is 26 not always a straightforward procedure and major obstacles are encountered when producing many eukaryotic 27 proteins and especially membrane proteins, linked to missing post-translational modifications, proteolysis and 28 aggregation. In this context, many conventional and unconventional eukaryotic hosts are under exploration 29 and development, but in some cases linked to complex culture media or processes. In this context, alternative 30 bacterial systems able to overcome some of the limitations posed by E. coli keeping the simplicity of 31 prokaryotic manipulation are currently emerging as convenient hosts for protein production. We have 32 comparatively produced a "difficult-to-express" human protein, the lysosomal enzyme alpha-galactosidase A 33 (hGLA) in E. coli and in the psychrophilic bacterium Pseudoalteromonas haloplanktis TAC125 cells (P. 34 halopanktis TAC125). While in E. coli the production of active hGLA was unreachable due to proteolytic 35 instability and/or protein misfolding, the expression of hGLA gene in P.halopanktis TAC125 allows obtaining 36 active enzyme. These results are discussed in the context of emerging bacterial systems for protein production 37Post-print of: Unzueta, Ugutz, et al. "Strategies for the production of difficult-to-express full-length eukaryotic proteins using microbial cell factories: production of human alpha-galactosidase

Lysosomal enzyme replacement therapies: Historical development, clinical outcomes, and future perspectives

“…Bacteria, in particular strains of Escherichia coli , have also been employed for this purpose, yet formation of protein aggregates as inclusion bodies and lack of post-translational modifications severely limit their applicability in producing recombinant enzymes for lysosomal ERT [227–231]. Alternative bacterial systems such as Pseudoalteromonas haloplanktis have been used to generate active α-galactosidase, but their uptake and effect have not been demonstrated yet [232]. …”

The pangenome of (Antarctic) Pseudoalteromonas bacteria: evolutionary and functional insights

“…Other signatures of cold adaptation are the psychrophiles-specific codon usage bias, that is involved in resistance to protein aging features involving asparagine cyclization and deamidation [10], and the high number of rRNA and tRNA genes, which might explain its translational efficiency even in cold condition [7]. This latter observation justifies an increasing use of Ph TAC125 in biotechnological applications, such as for the high quality production of recombinant eukaryotic proteins [11–13]. This prompted a comprehensive characterization of this strains, through the analysis of its complete genome [7], its proteome in different conditions [14, 15], detailed growth phenotypes in complex media [9, 11].…”

Improvement of Pseudoalteromonas haloplanktis TAC125 as a Cell Factory: IPTG-Inducible Plasmid Construction and Strain Engineering

“…Almost 20 years ago, our research group suggested the use of Ph TAC125 and its derived genetic tools for the setup of a novel cell factory working at low temperatures [ 14 ]. Till then, much evidence highlighted the notable skills of the Antarctic bacterium in the high quality production of human and/or eukaryotic complex proteins, reinforcing our original idea [ 2 , 3 , 5 , 6 , 11 , 34 , 35 ].…”

“…The ability to produce heterologous proteins with high yields is a prerequisite for the exploitation of a microorganism as a cell factory [ 33 ]. The psychrophilic bacterium Ph TAC125 represents a model as a non-conventional host for the production of difficult to express proteins in a soluble and active form [ 2 , 5 , 34 , 35 ]. In the present paper, the set of plasmids for controlled gene expression in Ph TAC125 and KrPL—a pMtBL deficient strain—has been expanded with the IPTG-inducible plasmid pP79.…”