The Pichia pastoris expression system is being used successfully for the production of various recombinant heterologous proteins. Recent developments with respect to the Pichia expression system have had an impact on not only the expression levels that can be achieved, but also the bioactivity of various heterologous proteins. We review here some of these recent developments, as well as strategies for reducing proteolytic degradation of the expressed recombinant protein at cultivation, cellular and protein levels. The problems associated with post-translational modifications performed on recombinant proteins by P. pastoris are discussed, including the effects on bioactivity and function of these proteins, and some engineering strategies for minimizing unwanted glycosylations. We pay particular attention to the importance of optimizing the physicochemical environment for efficient and maximal recombinant protein production in bioreactors and the role of process control in optimizing protein production is reviewed. Finally, future aspects of the use of the P. pastoris expression system are discussed with regard to the production of complex membrane proteins, such as G protein-coupled receptors, and the industrial and clinical importance of these proteins.

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Heterologous protein production using the Pichia pastoris expression system.

Homologous recombination applied to mouse embryonic stem (ES) cells has revolutionized the study of gene function in mammals. Although most often used to generate knockout mice, homologous recombination has also been applied in mouse ES cells allowed to differentiate in vitro. Homologous recombination is an essential technique if human ES cells are to fulfill their promise as a basic research tool. It also has important implications for ES cell-based transplantation and gene therapies. Significant differences between mouse and human ES cells have hampered the development of homologous recombination in human ES cells. High, stable transfection efficiencies in human ES cells have been difficult to achieve, and, in particular, electroporation protocols established for mouse ES cells work poorly in human ES cells. Also, in contrast to their murine counterparts, human ES cells cannot be cloned efficiently from single cells, making it difficult to screen for rare recombination events. Here we report an electroporation approach, based on the physical characteristics of human ES cells, that we used to successfully target HPRT1, the gene encoding hypoxanthine phosphoribosyltransferase-1 (HPRT1), and POU5F1, the gene encoding octamer-binding transcription factor 4 (Oct4; also known as POU domain, class 5, transcription factor 1 (POU5F1)).

Homologous recombination in human embryonic stem cells.

https://houptlab.org/courses/cmn/monoamines/Daubner2011.pdf

Tyrosine Hydroxylase and Regulation of Dopamine Synthesis

The α-synuclein gene is implicated in the pathogenesis of Parkinson's disease. Although α-synuclein function is uncertain, the protein has homology to the chaperone molecule 14-3-3. In addition, α-synuclein can bind to 14-3-3, and both α-synuclein and 14-3-3 bind to many of the same proteins. Because 14-3-3 binds to and activates tyrosine hydroxylase, the rate-limiting enzyme in dopamine (DA) biosynthesis, we explored whether α-synuclein also bound to tyrosine hydroxylase and influenced its activity. Immunoprecipitation revealed an interaction between α-synuclein and tyrosine hydroxylase in brain homogenates and MN9D dopaminergic cells. Colocalization of α-synuclein with tyrosine hydroxylase was confirmed by immunoelectron microscopy. To explore the consequences of the interaction, we measured the effect of recombinant α-synuclein on tyrosine hydroxylase activity in a cell-free system and observed a dose-dependent inhibition of tyrosine hydroxylase by α-synuclein. To measure the impact of α-synuclein on tyrosine hydroxylase in dopaminergic cells, we stably transfected MN9D cells with wild-type or A53T mutant α-synuclein. Overexpression of wild-type or A53T mutant α-synuclein did not significantly alter tyrosine hydroxylase protein levels in our stably transfected cells. However, overexpressing cell lines had significantly reduced tyrosine hydroxylase activity and a corresponding reduction in dopamine synthesis. The reduction in cellular dopamine levels was not caused by increased dopamine catabolism or dopamine efflux. These data suggest that α-synuclein plays a role in the regulation of dopamine biosynthesis, acting to reduce the activity of tyrosine hydroxylase. If so, a loss of soluble α-synuclein, by reduced expression or aggregation, could increase dopamine synthesis with an accompanying increase in reactive dopamine metabolites.

https://www.jneurosci.org/content/jneuro/22/8/3090.full.pdf

A Role for α-Synuclein in the Regulation of Dopamine Biosynthesis

14-3-3 proteins exert an extraordinarily widespread influence on cellular processes in all eukaryotes. They operate by binding to specific phosphorylated sites on diverse target proteins, thereby forcing conformational changes or influencing interactions between their targets and other molecules. In these ways, 14-3-3s ‘finish the job’ when phosphorylation alone lacks the power to drive changes in the activities of intracellular proteins. By interacting dynamically with phosphorylated proteins, 14-3-3s often trigger events that promote cell survival – in situations from preventing metabolic imbalances caused by sudden darkness in leaves to mammalian cell-survival responses to growth factors. Recent work linking specific 14-3-3 isoforms to genetic disorders and cancers, and the cellular effects of 14-3-3 agonists and antagonists, indicate that the cellular complement of 14-3-3 proteins may integrate the specificity and strength of signalling through to different cellular responses.

Dynamic interactions between 14-3-3 proteins and phosphoproteins regulate diverse cellular processes.

A consistent neurochemical abnormality in Parkinson's disease (PD) is degeneration of dopaminergic neurons in substantia nigra, leading to a reduction of striatal dopamine (DA) levels. As tyrosine hydroxylase (TH) catalyses the formation ofl-DOPA, the rate-limiting step in the biosynthesis of DA, the disease can be considered as a TH-deficiency syndrome of the striatum. Similarly, some patients with hereditaryl-DOPA-responsive dystonia, a neurological disorder with clinical similarities to PD, have mutations in the TH gene and decreased TH activity and/or stability. Thus, a logical and efficient treatment strategy for PD is based on correcting or bypassing the enzyme deficiency by treatment withl-DOPA, DA agonists, inhibitors of DA metabolism, or brain grafts with cells expressing TH. A direct pathogenetic role of TH has also been suggested, as the enzyme is a source of reactive oxygen species (ROS) in vitro and a target for radical-mediated oxidative injury. Recently, it has been demonstrated thatl-DOPA is effectively oxidized by mammalian TH in vitro, possibly contributing to the cytotoxic effects of DOPA. This enzyme may therefore be involved in the pathogenesis of PD at several different levels, in addition to being a promising candidate for developing new treatments of this disease.

Tyrosine hydroxylase and Parkinson's disease.

Recombinant human tyrosine hydroxylase (hTH1) was found to be phosphorylated by mitogen and stress-activated protein kinase 1 (MSK1) at Ser40 and by p38 regulated/activated kinase (PRAK) on Ser19. Phosphorylation by MSK1 induced an increase in Vmax and a decrease in Km for 6-(R)-5,6,7,8-tetrahydrobiopterin (BH4), while these kinetic parameters were unaffected as a result of phosphorylation by PRAK. Phosphorylation of both Ser40 and Ser19 induced a high-affinity binding of 14-3-3 proteins, but only the interaction of 14-3-3 with Ser19 increased the hTH1 activity. The 14-3-3 proteins also inhibited the rate of dephosphorylation of Ser19 and Ser40 by 82 and 36%, respectively. The phosphorylation of hTH1 on Ser19 caused a threefold increase in the rate of phosphorylation of Ser40. These studies provide new insights into the possible roles of stress-activated protein kinases in the regulation of catecholamine biosynthesis.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1471-4159.2002.01172.x

Regulation of tyrosine hydroxylase by stress-activated protein kinases

Tyrosine hydroxylase (TH) has been reported to require binding of 14-3-3 proteins for optimal activation by phosphorylation. We examined the effects of phosphorylation at Ser19, Ser31 and Ser40 of bovine TH and human TH isoforms on their binding to the 14-3-3 proteins BMH1/BMH2, as well as 14-3-3 zeta and a mixture of sheep brain 14-3-3 proteins. Phosphorylation of Ser31 did not result in 14-3-3 binding, however, phosphorylation of TH on Ser40 increased its affinity towards the yeast 14-3-3 isoforms BMH1/BMH2 and sheep brain 14-3-3, but not for 14-3-3 zeta. On phosphorylation of both Ser19 and Ser40, binding to the 14-3-3 zeta isoform also occurred, and the binding affinity to BMH1 and sheep brain 14-3-3 increased. Both phosphoserine-specific antibodies directed against the 10 amino acids surrounding Ser19 or Ser40 of TH, and the phosphorylated peptides themselves, inhibited the association between phosphorylated TH and 14-3-3 proteins. This was also found when heparin was added, or after proteolytic removal of the N-terminal 37 amino acids of Ser40-phosphorylated TH. Binding of BMH1 to phosphorylated TH decreased the rate of dephosphorylation by protein phosphatase 2A, but no significant change in enzymatic activity was observed in the presence of BMH1. These findings further support a role for 14-3-3 proteins in the regulation of catecholamine biosynthesis and demonstrate isoform specificity for both TH and 14-3-3 proteins.

Interaction of phosphorylated tyrosine hydroxylase with 14-3-3 proteins: evidence for a phosphoserine 40-dependent association.

Expression and purification of human tryptophan hydroxylase from Escherichia coli and Pichia pastoris

Tetrahydropterins are obligatory cofactors for tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine biosynthesis. A series of synthetic analogues of 6(R)-L-erythro-5,6,7, 8-tetrahydrobiopterin (BH(4)) with different substituents in positions C2, N3, C4, N5, C6, C7, and N8 on the ring were used as active site probes for recombinant human TH. The enzyme tolerates rather bulky substituents at C6, as seen by the catalytic efficiency (V(max)/K(m)) and the coupling efficiency (mol of L-DOPA produced/mol of tetrahydropterin oxidized) of the cofactors. Substitutions at C2, C4, N5, and N8 abolish the cofactor activity of the pterin analogues. Molecular docking of BH(4) into the crystal structure of the catalytic domain of ligand-free rat TH results in complexes in which the pteridine ring pi-stacks with Phe300 and the N3 and the amino group at C2 hydrogen bonds with Glu332. The pteridine ring also establishes interactions with Leu294 and Gln310. The distance between C4a in the pteridines and the active site iron was 4.2 +/- 0.5 A for the ensemble of docked conformers. Docking of BH(4) analogues into TH also shows that the most bulky substituents at C6 can be well-accommodated within the large hydrophobic pocket surrounded by Ala297, Ser368, Tyr371, and Trp372, without altering the positioning of the ring. The pterin ring of 7-BH(4) shows proper stacking with Phe300, but the distance between the C4a and the active site iron is 0.6 A longer than for bound BH(4), a finding that may be related to the high degree of uncoupling observed for 7-BH(4).

Karen Toska

Papers

Tyrosine hydroxylase and Parkinson's disease.

Regulation of tyrosine hydroxylase by stress-activated protein kinases

Interaction of phosphorylated tyrosine hydroxylase with 14-3-3 proteins: evidence for a phosphoserine 40-dependent association.

Expression and purification of human tryptophan hydroxylase from Escherichia coli and Pichia pastoris

A kinetic and conformational study on the interaction of tetrahydropteridines with tyrosine hydroxylase.