Optimization of Pichia pastoris Expression System for High-Level Production of Margatoxin

Margatoxin (MgTx) is a high-affinity blocker of voltage-gated potassium (Kv) channels. It inhibits Kv1.1–Kv1.3 ion channels in picomolar concentrations. This toxin is widely used to study physiological function of Kv ion channels in various cell types, including immune cells. Isolation of native MgTx in large quantities from scorpion venom is not affordable. Chemical synthesis and recombinant production in Escherichia coli need in vitro oxidative refolding for proper disulfide bond formation, resulting in a very low yield of peptide production. The Pichia pastoris expression system offers an economical approach to overcome all these limitations and gives a higher yield of correctly refolded recombinant peptides. In this study, improved heterologous expression of recombinant MgTx (rMgTx) in P. pastoris was obtained by using preferential codons, selecting the hyper-resistant clone against Zeocin, and optimizing the culturing conditions. About 36 ± 4 mg/L of >98% pure His-tagged rMgTx (TrMgTx) was produced, which is a threefold higher yield than has been previously reported. Proteolytic digestion of TrMgTx with factor Xa generated untagged rMgTx (UrMgTx). Both TrMgTx and UrMgTx blocked the Kv1.2 and Kv1.3 currents (patch-clamp) (Kd for Kv1.2 were 64 and 14 pM, and for Kv1.3, 86 and 50 pM, respectively) with comparable potency to the native MgTx. The analysis of the binding kinetics showed that TrMgTx had a lower association rate than UrMgTx for both Kv1.2 and Kv1.3. The dissociation rate of both the analogues was the same for Kv1.3. However, in the case of Kv1.2, TrMgTx showed a much higher dissociation rate with full recovery of the block than UrMgTx. Moreover, in a biological functional assay, both peptides significantly downregulated the expression of early activation markers IL2R and CD40L in activated CD4+ TEM lymphocytes whose activation was Kv1.3 dependent. In conclusion, the authors report that the Pichia expression system is a powerful method to produce disulfide-rich peptides, the overexpression of which could be enhanced noticeably through optimization strategies, making it more cost-effective. Since the presence of the His-tag on rMgTx only mildly altered the block equilibrium and binding kinetics, recombinant toxins could be used in ion channel research without removing the tag and could thus reduce the cost and time demand for toxin production.

Production of a Recombinant Single-Domain Antibody for Gluten Detection in Foods Using the Pichia pastoris Expression System

The detection of gluten in foodstuffs has become a growing concern in food allergen management as a result of the high ratio of population sensitive to the main gluten-containing cereals. In this study, a promising single-domain antibody previously isolated by phage display (dAb8E) was produced in Pichia pastoris resulting in high levels of the antibody fragment expression (330 mg/L). The purified dAb8E was proved to specifically bind to gluten proteins from wheat, barley and rye, exhibiting no cross reaction to other heterologous species. The dynamic range of the sandwich enzyme-linked immunosorbent assay (ELISA) covered 0.1 to 10 µg/mL of gliadin, reaching a limit of detection of 0.12 µg/mL. When experimental binary mixtures of the target cereals were analyzed, the limit of detection was 0.13 mg/g, which would theoretically correspond to gluten concentrations of approximately 13 mg/kg. Finally, thirty commercially available food products were analyzed by means of the developed assay to further confirm the applicability of the dAb8E for gluten determination. The proposed methodology enabled the generation of a new gluten-specific nanobody which could be used to guarantee the appropriate labelling of gluten-free foods.

Molecular expression of a recombinant thermostable bacterial amylase from Geobacillus stearothermophilus SR74 using methanol-free Meyerozyma guilliermondii strain SO yeast system

α-Amylase, which was isolated from Geobacillus stearothermophilus SR74, has shown its potential to be used in industrial applications. However, its expression in the Pichia pastoris expression system with the alcohol oxidase 1 promoter (PAOX1) requires high methanol consumption and is time-consuming. This study aimed to express SR74 α-amylase in an alternative yeast system, using Meyerozyma guilliermondii strain SO, which was isolated from a spoiled orange (SO) under the regulation of a formaldehyde dehydrogenase promoter (PFLD). Qualitative screening showed that strain SO possessed a native amylase grown on YPD-starch plate at 30 °C. The recombinant SR74 α-amylase was further quantified and validated using the Western blot test. It was confirmed that SR74 α-amylase was expressed by strain SO extracellularly with a size of 59 kDa. Optimization in a shake flask showed that the recombinant SR74 α-amylase, which was regulated by PFLD, was successfully produced (26 U/mL) without any external inducer in the YPT medium after 24 h of cultivation. In conclusion, strain SO was able to produce SR74 amylase without methanol in one-fifth the fermentation time of P. pastoris. Further optimization of the expression may be done to improve the yield, as this methanol-free host is still underexplored.

Characterization of a highly stable α-galactosidase from thermophilic Rasamsonia emersonii heterologously expressed in a modified Pichia pastoris expression system

Background Structurally stable α-galactosidases are of great interest for various biotechnological applications. More thermophilic α-galactosidases with high activity and structural stability have therefore to be mined and characterized. On the other hand, few studies have been performed to prominently enhance the AOX1 promoter activity in the commonly used Pichia pastoris system, in which production of some heterologous proteins are insufficient for further study. Results ReGal2 encoding a thermoactive α-galactosidase was identified from the thermophilic (hemi)cellulolytic fungus Rasamsonia emersonii. Significantly increased production of ReGal2 was achieved when ReGal2 was expressed in an engineered Pastoris pichia expression system with a modified AOX1 promoter and simultaneous fortified expression of Mxr1 that is involved in transcriptionally activating AOX1. Purified ReGal2 exists as an oligomer and has remarkable thermo-activity and thermo-tolerance, exhibiting maximum activity of 935 U/mg towards pNPGal at 80 °C and retaining full activity after incubation at 70 °C for 60 h. ReGal2 is insensitive to treatments by many metal ions and exhibits superior tolerance to protein denaturants. Moreover, ReGal2 efficiently hydrolyzed stachyose and raffinose in soybeans at 70 °C in 3 h and 24 h, respectively. Conclusion A modified P. pichia expression system with significantly enhanced AOX1 promoter activity has been established, in which ReGal2 production is markedly elevated to facilitate downstream purification and characterization. Purified ReGal2 exhibited prominent features in thermostability, catalytic activity, and resistance to protein denaturants. ReGal2 thus holds great potential in relevant biotechnological applications.