Heterologous expression of the mammalian sodium-nucleobase transporter rSNBT1 in Leishmania tarentolae

Recombinant expression systems for mammalian membrane transport proteins are often limited by insufficient yields to support structural studies, inadequate post-translational processing and problems related with improper membrane targeting or cytotoxicity. Use of alternative expression systems and optimization of expression/purification protocols are constantly needed. In this work, we explore the applicability of the laboratory strain LEXSY of the ancient eukaryotic microorganism Leishmania tarentolae as a new expression system for mammalian nucleobase permeases of the NAT/NCS2 (Nucleobase-Ascorbate Transporter/Nucleobase-Cation Symporter-2) family. We achieved the heterologous expression of the purine-pyrimidine permease rSNBT1 from Rattus norvegicus (tagged at C-terminus with a red fluorescent protein), as confirmed by confocal microscopy and biochemical analysis of the subcellular fractions enriched in membrane proteins. The cDNA of rSNBT1 has been subcloned in a pLEXSY-sat-mrfp1vector and used to generate transgenic L. tarentolae-rsnbt1-mrfp1 strains carrying the pLEXSY-sat-rsnbt1-mrfp1 plasmid either episomally or integrated in the chromosomal DNA. The chimeric transporter rSNBT1-mRFP1 is targeted to the ER and the plasma membrane of the L. tarentolae promastigotes. The transgenic strains are capable of transporting nucleobases that are substrates of rSNBT1 but also of the endogenous L. tarentolae nucleoside/nucleobase transporters. A dipyridamole-resistant Na⁺-dependent fraction of uptake is attributed to the exogenously expressed rSNBT1.     

Developmental expression of the murine Prl-1 protein tyrosine phosphatase gene

The expression of the murine Prl-1 protein tyrosine phosphatase gene was examined in normal embryos from E10.5 through E18.5. Prl-1 mRNA was detected in the brain, neural tube, and dorsal root ganglia, and in several non-neuronal tissues, including the skeletal system. Heart and skeletal muscle were consistently negative. At E13.5, Prl-1 was expressed in the condensing prechondrogenic cells of the vertebrae, whereas at E18.5, Prl-1 mRNA was localized to the hypertrophic chondrocytes. The dynamic expression of Prl-1 during cartilage differentiation may suggest a functional role in skeletal development.     

Down-regulation of protein tyrosine phosphatase gene expression in lactating mouse mammary gland

Detailed analysis of protein tyrosine phosphatase (PTP) expression in mouse mammary gland and mammary epithelial cells using a set of degenerate primers corresponding to the PTP core domain sequence revealed the presence of 16 different receptor-type and intracellular PTPs. Northern blot and RT-PCR analyses revealed that some PTPs were up-regulated during gestation, suggesting that these enzymes are involved in development of mammary gland. However, expression of most PTPs dramatically decreased during lactation, whereas the beta-casein gene expression was increased and remained at a high level. At the involution stage after weaning, most PTPs were up-regulated and their expression returned almost to the virgin level. Such up-regulation was also induced by forced weaning in lactating mother mice. These results suggest the possible contribution of PTPs to the development, involution, and remodeling of mammary gland and their possible inhibitory action on maintaining high expression of milk genes during lactation.     

Heterologous protein production using euchromatin-containing expression vectors in mammalian cells

Upon stable cell line generation, chromosomal integration site of the vector DNA has a major impact on transgene expression. Here we apply an active gene environment, rather than specified genetic elements, in expression vectors used for random integration. We generated a set of Bacterial Artificial Chromosome (BAC) vectors with different open chromatin regions, promoters and gene regulatory elements and tested their impact on recombinant protein expression in CHO cells. We identified the Rosa26 BAC as the most efficient vector backbone showing a nine-fold increase in both polyclonal and clonal production of the human IgG-Fc. Clonal protein production was directly proportional to integrated vector copy numbers and remained stable during 10 weeks without selection pressure. Finally, we demonstrated the advantages of BAC-based vectors by producing two additional proteins, HIV-1 glycoprotein CN54gp140 and HIV-1 neutralizing PG9 antibody, in bioreactors and shake flasks reaching a production yield of 1 g/l.     

Cloning and expression of a yeast protein tyrosine phosphatase.

To study the regulation of tyrosine phosphorylation/dephosphorylation in Saccharomyces cerevisiae, a protein tyrosine phosphatase (PTPase) was cloned by the polymerase chain reaction (PCR). Conserved amino acid sequences within the mammalian PTPases were used to design primers which generated a yeast PCR fragment. The sequence of the PCR fragment encoded a protein with homology to the mammalian PTPases. The PCR fragment was used to identify the yeast PTP1 gene which has an open reading frame encoding a 335-amino acid residue protein. This yeast PTPase shows 26% sequence identity to the rat PTPase, although highly conserved residues within the mammalian enzymes are invariant in the yeast protein. The yeast PTP1 is physicallt linked to the 5'-end of a heat shock gene SSB1. This yeast PTP1 gene was expressed in Escherichia coli and obtained in a highly purified form by a single affinity chromatography step. The recombinant yeast PTPase hydrolyzed phosphotyrosine containing substrates approximately 1000 times faster than a phosphoserine containing substrate. Gene disruption of yeast PTP1 has no visible effect on vegetative growth.     

Differential expression of a novel murine non-receptor protein tyrosine phosphatase during differentiation of P19 embryonal carcinoma cells.

Protein phosphorylation on tyrosine residues is one of the major mechanisms of cell signal transduction and is regulated by protein tyrosine kinases and protein tyrosine phosphatases. Here we report the molecular cloning of an additional member of the protein tyrosine phosphatase-family from differentiated murine P19 embryonal carcinoma cells. This non-receptor protein tyrosine phosphatase, P19-PTP, does not contain regulatory sequences, homologous to the ones found in other non-receptor PTPases. P19-PTP is differentially expressed during in vitro differentiation of P19 EC cells, in that P19-PTP mRNA could only be detected in embryoid bodies, derived from P19 cells.     

Heterologous expression of a mammalian epithelial sodium channel in yeast

The alpha and beta subunits of the amiloride-sensitive rat epithelial sodium channel (alpha beta ENaC) were expressed in the yeast Saccharomyces cerevisiae. We used a combination of yeast strains, including a mutant in the secretory pathway (sec6), and Western blotting techniques, to show that alpha beta ENaC was synthesized and targeted through the secretory system to the plasma membrane. Yeasts expressing alpha beta ENaC were more sensitive to salt than the parent strain. In addition, amiloride, a specific blocker of ENaC, was found to suppress salt sensitivity in the yeast strain expressing alpha beta ENaC.     

Temporal variations in protein tyrosine phosphatase activity during cell proliferation and differentiation

Cellular oscillations in total extractable protein content and protein tyrosine phosphatase activity were analysed in proliferating and differentiating human acute promyelocytic leukaemic (HL-60) cell extracts. Differentiation was induced along the granulocytic pathway using all-trans retinoic acid (ATRA). High frequency rhythms with distinct, well defined waveforms of varying amplitude were observed for both the total protein content and for the enzyme activity of protein tyrosine phosphatase (PTP). Linear correlation analysis showed that there was no obvious relationship between the protein content and the corresponding PTP activity, suggesting that the periodic variations between these two components are relatively independent of each other. ATRA significantly altered the characteristics of the rhythms with respect to the period and amplitude and had a dampening effect on the oscillatory pattern of PTP activity. Modulation of such characteristics may be of significance with respect to the regulation of the differentiation processes and the possible reversal of transformation.     

HD-PTP: A novel protein tyrosine phosphatase gene on human chromosome 3p21.3

A human cDNA encoding a novel protein tyrosine phosphatase has been isolated. The phosphatase has unique features in its domain structure: a "Zn-hand" domain containing several SH3-binding motifs, a tyrosine phosphatase domain, a C-terminal PEST motif, and an N-terminal domain similar to yeast BRO1, an apoptosis-related mammalian AIP1 and to a RHO-binding protein, Rhophilin. The gene is located at chromosome 3p21.3, an area frequently deleted in many types of cancer, especially within the functionally defined narrow region. The gene may be a human homolog of the rat PTP-TD14 gene reported by others, which can suppress H-ras-mediated transformation. We identified a hemizygous missense mutation in a lung cancer cell line. Thus, the phosphatase gene may be a candidate for one of the tumor suppressor genes located on 3p21.3.     

Periodinates: a new class of protein tyrosine phosphatase inhibitors

A series of periodinates has been synthesized and tested as protein tyrosine phosphatase substrates. Their potency is comparable to or higher than that of vanadates but much lower than that of peroxovanadates.     

Targeted mutagenesis and genetic analysis of a Drosophila receptor-linked protein tyrosine phosphatase gene

Several Drosophila receptor-linked protein tyrosine phosphatases (R-PTPs) are selectively expressed on axons of the developing embryonic central nervous system. The extracellular domains of these axonal R-PTPs are homologous to neural adhesion molecules. Thus, R-PTPs may directly couple cell recognition to signal transduction via control of tyrosine phosphorylation. To examine the function of these molecules during nervous system development, we wished to generate mutations in R-PTP genes. It was unclear whether a mutation in a single R-PTP gene would confer lethality, however, because the similarities in sequence and expression pattern between the axonal R-PTPs suggest that they may have partially redundant functions. To circumvent this problem, we developed a directed mutagenesis strategy based on local transposition of P elements, and used this approach to isolate a null mutation in the DPTP99A gene. This strategy, which we describe in detail here, should be applicable to any Drosophila gene within a lettered division of an appropriately marked P element. Flies lacking DPTP99A expression are viable and fertile, and we have been unable to detect any alterations in the embryonic nervous system of DPTP99A embryos using a variety of antibody markers.     

Heterologous gene expression in a membrane-protein-specific system.

We have constructed an expression system for heterologous proteins which uses the molecular machinery responsible for the high level production of bacteriorhodopsin in Halobacterium salinarum. Cloning vectors were assembled that fused sequences of the bacterio-opsin gene (bop) to coding sequences of heterologous genes and generated DNA fragments with cloning sites that permitted transfer of fused genes into H. salinarum expression vectors. Gene fusions include: (i) carboxyl-terminal-tagged bacterio-opsin; (ii) a carboxyl-terminal fusion with the catalytic subunit of the Escherichia coli aspartate transcarbamylase; (iii) the human muscarinic receptor, subtype M1; (iv) the human serotonin receptor, type 5HT2c; and (v) the yeast alpha mating factor receptor, Ste2. Characterization of the expression of these fusions revealed that the bop gene coding region contains previously undescribed molecular determinants which are critical for high level expression. For example, introduction of immunogenic and purification tag sequences into the C-terminal coding region significantly decreased bop gene mRNA and protein accumulation. The bacteriorhodopsin-aspartate transcarbamylase fusion protein was expressed at 7 mg per liter of culture, demonstrating that E. coli codon usage bias did not limit the system's potential for high level expression. The work presented describes initial efforts in the development of a novel heterologous protein expression system, which may have unique advantages for producing multiple milligram quantities of membrane-associated proteins.     

Protein tyrosine phosphatase gene expression analysis in Swiss 3T3 fibroblasts

The aim of this study was to identify protein tyrosine phosphatases (PTPs) expressed in Swiss 3T3 fibroblasts and to examine their expression levels as well as to characterize quantitative aspects of RT-PCR based on degenerate deoxyoligonucleotides. By using an RT-PCR assay based on degenerate deoxyoligonucleotide primers, expression of mRNAs for two cytoplasmic- and six transmembrane-type PTPs in Swiss 3T3 cells was detected. The sequences of two of them are new. Among nine analyzed PTPs expressed to widely varied extends, only three have mRNA levels high enough to be seen on Northern blots with 10 µg of total RNA per lane. The frequencies with which the examined PTPs are represented among the PCR amplification products, correlate stronger with the primer fidelity, defined as the number of mismatches between the primer- and the cDNA target-sequences, rather than with the PTP expression levels. In conclusion, an RT-PCR assay based on degenerate primers can be successfully used to sample the expressed PTPs and to identify new members of this gene family. However, reliable quantification of their mRNA levels can only be achieved using the classical approaches, like Northern, RNase protection assay or non-degenerate quantitative RT-PCR.