Genome-wide identification and in-silico analysis of papain-family cysteine protease encoding genes in Tetrahymena thermophila.

Tetrahymena thermophila is a promising host for recombinant protein production, but its utilization in biotechnology is mostly limited due to the presence of intracellular and extracellular papain-family cysteine proteases (PFCPs). In this study, we employed bioinformatics approaches to investigate the T. thermophila PFCP genes and their encoded proteases (TtPFCPs), the most prominent protease family in the genome. Results from the multiple sequence alignment, protein modeling, and conserved motif analyses revealed that all TtPFCPs showed considerably high homology with mammalian cysteine cathepsins and contained conserved amino acid motifs. The total of 121 TtPFCP-encoding genes, 14 of which were classified as non-peptidase homologs, were found. Remaining 107 true TtPFCPs were divided into four distinct subgroups depending on their homology with mammalian lysosomal cathepsins: cathepsin L-like (TtCATLs), cathepsin B-like (TtCATBs), cathepsin C-like (TtCATCs), and cathepsin X-like (TtCATXs) PFCPs. The majority of true TtPFCPs (96 out of the total) were in TtCATL-like peptidase subgroup. Both phylogenetic and chromosomal localization analyses of TtPFCPs supported the hypothesis that TtPFCPs likely evolved through tandem gene duplication events and predominantly accumulated on micronuclear chromosome 5. Additionally, more than half of the identified TtPFCP genes are expressed in considerably low quantities compared to the rest of the TtPFCP genes, which are expressed at a higher level. However, their expression patterns fluctuate based on the stage of the life cycle. In conclusion, this study provides the first comprehensive in-silico analysis of TtPFCP genes and encoded proteases. The results would help designing an effective strategy for protease knockout mutant cell lines to discover biological function and to improve the recombinant protein production in T. thermophila.

Recombinant production of hormonally active human insulin from pre-proinsulin by Tetrahymena thermophila.

Alternative cell factories, such as the unicellular ciliate eukaryotic Tetrahymena thermophila, may be required for the production of protein therapeutics that are challenging to produce in conventional expression systems. T. thermophila (Tt) can secrete proteins with the post-translational modifications necessary for their function in humans. In this study, we tested if T. thermophila could process the human pre-proinsulin to produce hormonally active human insulin (hINS) with correct modifications. Flask and bioreactor culture of T. thermophila were used to produce the recombinant Tt-hINS either with or without an affinity tag from a codon-adapted pre-proinsulin sequence. Our results indicate that T. thermophila can produce a 6 kDa Tt-hINS monomer with the appropriate disulfide bonds after removal of the human insulin signal sequence or endogenous phospholipase A signal sequence, and the C-peptide of the human insulin. Additionally, Tt-hINS can form 12 kDa dimeric, 24 kDa tetrameric, and 36 kDa hexameric complexes. Tt-hINS-sfGFP fusion protein was localized to the vesicles within the cytoplasm and was secreted extracellularly. Assessing the affinity-purified Tt-hINS activity using the in vivo T. thermophila extracellular glucose drop assay, we observed that Tt-hINS induced a significant reduction (approximately 21 %) in extracellular glucose levels, indicative of its functional insulin activity. Our results demonstrate that T. thermophila is a promising candidate for the pharmaceutical and biotechnology industries as a host organism for the production of human protein drugs.

The in vivo Tetrahymena thermophila extracellular glucose drop assay for characterization of mammalian insulin activity.

Insulin activity is generally determined by an in vivo rabbit blood glucose drop assay in research and industriel laboratories. The humane experimental techniques imply the use of alternative invertebrate organisms in place of animals, known as replacement rule of the 3Rs. In this study, we report an alternative in vivo extracellular glucose drop assay using unicellular invertebrate Tetrahymena thermophila to replace the use of rabbit and mouse. This assay has four major steps; growing cells, starving cells, treatment of cells and measurement of glucose drop. In this assay, 0.2 mg/ml of human, porcine and bovine insulins dropped extracellular glucose level to 16%, 14% and 12%, respectively in ten minutes. In addition, mammalian insulins respectively increased the cell area about 19%, 15%, and 16% at 6th hour with statistically significant effect on the cell growth, but not in the cell viability. The results showed that the in vivo Tetrahymena thermophila extracellular glucose drop assay could be used as an alternative assay to replace the mouse or the rabbit insulin blood glucose drop assay.

Characterization and use of Tetrahymena thermophila artificial chromosome 2 (TtAC2) constructed by biomimetic of macronuclear rDNA minichromosome.

Efficient expression vectors for unicellular ciliate eukaryotic Tetrahymena thermophila are still needed in recombinant biology and biotechnology applications. Previously, the construction of the T. thermophila Macronuclear Artificial Chromosome 1 (TtAC1) vector revealed additional needs for structural improvements such as better in vivo stability and maintenance as a recombinant protein expression platform. In this study, we designed an efficiently maintained artificial chromosome by biomimetic of the native macronuclear rDNA minichromosome. TtAC2 was constructed by sequential cloning of subtelomeric 3'NTS region (1.8 kb), an antibiotic resistance gene cassette (2 kb neo4), a gene expression cassette (2 kb TtsfGFP), rDNA coding regions plus a dominant C3 origin sequence (10.3 kb), and telomeres (2.4 kb) in a pUC19 backbone plasmid (2.6 kb). The 21 kb TtAC2 was characterized using fluorescence microscopy, qPCR, western blot and Southern blot after its transformation to vegetative T. thermophila CU428.2 strain, which has a recessive B origin allele. All experimental data show that circular or linear forms of novel TtAC2 were maintained as free replicons in T. thermophila macronucleus with or without antibiotic treatment. Notably, TtAC2 carrying strains expressed a TtsfGFP marker protein, demonstrating the efficacy and functionality of the protein expression platform. We show that TtAC2 is functionally maintained for more than two months, and can be efficiently used in recombinant DNA, and protein production applications.

Construction and dynamic characterization of a Tetrahymena thermophila macronuclear artificial chromosome.

Artificial chromosomes were previously generated for use in bacteria, protists, yeast and human cells. A Tetrahymena thermophila artificial chromosome could serve as a versatile platform to study diverse aspects of Tetrahymena biology and beyond. Here, we placed a C3-type rDNA replication origin and telomere sequences from T. thermophila into a pNeo4 vector, producing the first T. thermophila macronuclear artificial chromosome (TtAC1). Circular or linear forms of TtAC1 can be stably transformed into both vegetative and conjugative T. thermophila cells. Linear TtAC1 was stably double in copy number under antibiotic selection, but its copy number was dropping without antibiotic selection pressure. Southern blot, Real-Time PCR and E. coli retransformation analyses together showed that TtAC1 vector did not integrate into the macronuclear genome, and was maintained as a linear or a circular chromosome in T. thermophila macronucleus under antibiotic selection. The use of TtAC1 for recombinant protein production was demonstrated by western blot analysis of a secreted 27 kDa TtsfGFP-12XHis protein. We present the first macronuclear artificial chromosome with species-specific chromosomal elements for use in T. thermophila studies and to aid broad recombinant biotechnology applications.

Genome-wide analysis of the Tetrahymena thermophila glutathione S-transferase gene superfamily.

The ciliate Tetrahymena thermophila has a rapid response to detoxify xenobiotics, which presents opportunity to study the diversification of Glutathione S-Transferase superfamily. In-silico identification of putative GST genes were resulted with 70 GST genes; 49 TtGSTmu, 7 TtGSTomega, 5 TtGSTtheta, 2 TtGSTzeta, 4 TtMAPEG and 3 TtEF1G. TtGST superfamily has short intron carrying or intronless genes. The most expressed mRNAs of TtGST are limited to 4 members at all life stages. TtGST genes are widely distributed to all five micronuclear chromosomes with the highest diversified members from different classes in chromosome 4. The clustering and the orientation of some TtGSTs in the T. thermophila genome give clues about the recent gene duplication. Analysis of GSH affinity-purified GST proteins with Western blot and activity assay showed GST activity carrying purified TtGST populations. In conclusion, the enhanced genome capacity of TtGST superfamily may have evolved through improved GST enzymatic activity.