Use of the KlADH3 promoter for the quantitative production of the murine PDE5A isoforms in the yeast Kluyveromyces lactis

Abstract

Background: Phosphodiesterases (PDE) are a superfamily of enzymes that hydrolyse cyclic nucleotides (cAMP/cGMP), signal molecules in transduction pathways regulating crucial aspects of cell life. PDEs regulate the intensity and duration of the cyclic nucleotides signal modulating the downstream biological effect. Due to this critical role associated with the extensive distribution and multiplicity of isozymes, the 11 mammalian families (PDE1 to PDE11) constitute key therapeutic targets. PDE5, one of these cGMP-specific hydrolysing families, is the molecular target of several well known drugs used to treat erectile dysfunction and pulmonary hypertension. Kluyveromyces lactis, one of the few yeasts capable of utilizing lactose, is an attractive host alternative to Saccharomyces cerevisiae for heterologous protein production. Here we established K. lactis as a powerful host for the quantitative production of the murine PDE5 isoforms.

Results: Using the promoter of the highly expressed KlADH3 gene, multicopy plasmids were engineered to produce the native and recombinant Mus musculus PDE5 in K. lactis. Yeast cells produced large amounts of the purified A1, A2 and A3 isoforms displaying K_m, V_max and Sildenafil inhibition values similar to those of the native murine enzymes. PDE5 whose yield was nearly 1 mg/g wet weight biomass for all three isozymes (30 mg/L culture), is well tolerated by K. lactis cells without major growth deficiencies and interferences with the endogenous cAMP/cGMP signal transduction pathways.

Conclusions: To our knowledge, this is the first time that the entire PDE5 isozymes family containing both regulatory and catalytic domains has been produced at high levels in a heterologous eukaryotic organism. K. lactis has been shown to be a very promising host platform for large scale production of mammalian PDEs for biochemical and structural studies and for the development of new specific PDE inhibitors for therapeutic applications in many pathologies.

Keywords: KlADH3 promoter; Kluyveromyces lactis; Multicopy plasmids; Murine PDE5; Sildenafil; cGMP.

Fig. 1

Kluyveromyces lactis in-gel native ADH pattern. Cells were grown for 2–3 days in YP medium supplemented with the carbon sources at 2%, unless stated in the text. Extracts from these cultures fractioned on native PAGE were stained for ADH. The migrating positions of ADH isoforms are reported on the left. The  < >  symbols indicate switch of substrate after 2 days of growth

Fig. 2

Vectors used for the expression of Pde5a genes. a N-terminal variants of PDE5A1, A2 and A3 isoforms. b Plasmid PDE5A inserts. c Map of the pYG137/1 used for the expression of PDE5A1 and recombinant derivatives. pKD1 sequences in grey. α-peptide/3XFlag schemes are not in scale with those of PDE5A

Fig. 3

Analysis of ADH and PDE5 activities from cellular extracts. a In-gel native ADH pattern from the wild type (WT) and pα-PDE5A1 transformed strain. Cells were grown in YPG or YPD medium for 1–5 days. The migrating position of ADH isoforms is reported on the left. b cGMP hydrolysing activities from extracts of WT and pα-PDE5A1 transformed strain. Extracts were obtained from YPG-grown cultures of a. c Western blot of PDE5A1 from the 2–4 days YPD- YPG-grown cultures extracts of a (lanes 7–12) and extracts from YP lactate or acetate cultures. d cGMP hydrolysing activities with/without 200 nM Sildenafil (sild). Soluble (s) and pellet fraction (p) were from the 4 days YPG-grown cultures extracts of a. e Western blot of PDE5A1 from the 4 days YPG-grown cellular extracts and medium supernatants of a concentrated 20 times. N18TG2 (N18) murine neuroblastoma cell line extract is used as PDE5 standard. Each lane contains 10 μg of protein extract for ADH analysis, 30 μg for western blot of PDE or the concentrated equivalent of 0.3 mL of medium supernatant. Arrows indicate the migrating position of PDE5 bands

Fig. 4

Native PAGE-migrating properties of cAMP/cGMP hydrolysing activities from WT and pα-PDE5A1 culture extracts. The activities of both extracts were determined in CPM (counts per minute) in the sequentially-sliced (1–20) migrated extracts shown in the Coomassie-stained gel insert. Extracts were obtained from cells grown in YPG medium for 4 days

Fig. 5

Analyses of PDE5 activities from cellular extracts. a SDS-PAGE analysis by Coomassie-staining and western blot of extracts from the WT and the pPDE5A1 transformed strain. b cGMP hydrolysing activities from the soluble (s) and pellet fraction (p) extracts of a. c SDS-PAGE analysis by Coomassie-staining of extracts from the pα-PDE5A1, the pPDE5A1 and the p3XFlagPDE5A1 transformed strains. d Determination of cGMP hydrolysing activities from the extracts of c. Cultures were grown in YPG medium for 4 days. Each lane contains 10 μg of protein extract. The arrows indicate the bands of PDE5A1

Fig. 6

Growth curves and viability of PDE5-transformed strains. a Growth curve of WT, pα-PDE5A1 and p3XFlagPDE5A1 transformed strains. Cultures were grown in YPG medium for 4 days and OD₆₀₀ determined at time intervals. Each value is the average of three independent determinations with a standard deviation comprised between 4 and 13%. b Cell viability of cultures of A was determined at the end of 4 days of growth. Cell cultures were diluted to 10⁸ cell/mL and the viability expressed as % of those forming colonies on YPD plates. c Western blot of PDE5A1 from the 2–4 days YPD-YPG-grown cultures extracts of the p3XFlagPDE5A1 strain. Each lane contains 4 μg of proteins. S. cerevisiae Pgk1 antibody was used as loading standard

Fig. 7

Coomassie-stained SDS-PAGE analysis of the 3XFlagPDE5 isoforms purification processes. The figure reports the whole protein extracts (WE), the flow-through (FT) and the eluted fractions (I, II and III) of the A1 (lanes 1–8) and A2 (lanes 9–16) isoforms purified by an ANTI-FLAG M2 affinity chromatography column. The figure also reports the different MW of the three purified isoforms (lanes 17–19). WE was from 100 mL of YPG cultures grown for 4 days. BSA standards were used for the quantification

Fig. 8

Biochemical properties of the purified 3XFlagPDE5A1 protein. a Eadie-Hofstee representation plot of cGMP hydrolysis. b Sildenafil inhibition curve. c Native PAGE gel shift by pre-incubation of 3XFlagPDE5A1 with Sildenafil. The purified 3XFlagPDE5A1 protein (1 μg) was incubated for 15′ at 30 °C with the inhibitor, fractioned on gel and stained with Coomassie