Pastoris Culture Research Articles

Spider venom neurotoxin based bioinsecticides: A novel bioactive for the control of the Asian citrus psyllid Diaphorina citri (Hemiptera)

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is a key vector of the phloem-limited bacteria Candidatus Liberibacter asiaticus (CLas) associated with huanglongbing (HLB), the most serious and currently incurable disease of citrus worldwide. Here we report the first investigation into the potential use of a spider venom-derived recombinant neurotoxin, ω/κ-HxTx-Hv1h (hereafter HxTx-Hv1h) when delivered alone or when fused to snowdrop lectin (Galanthus nivalis agglutinin; GNA) to control D. citri. Proteins, including GNA alone, were purified from fermented transformed yeast Pichia pastoris cultures. Recombinant HxTx-Hv1h, HxTx-Hv1h/GNA and GNA were all orally toxic to D. citri, with Day 5 median lethal concentrations (LC50) derived from dose-response artificial diet assays of 27, 20 and 52 μM, respectively. Western analysis of whole insect protein extracts confirmed that psyllid mortality was attributable to protein ingestion and that the fusion protein was stable to cleavage by D. citri proteases. When applied topically (either via droplet or spray) HxTx-Hv1h/GNA was the most effective of the proteins causing >70 % mortality 5 days post treatment, some 2 to 3-fold higher levels of mortality as compared to the toxin alone. By contrast, no significant mortality or phenotypic effects were observed for bumble bees (Bombus terrestris L.) fed on the recombinant proteins in acute toxicity assays. This suggests that HxTx-Hv1h/GNA has potential as a novel bioinsecticide for the management of D. citri offering both enhanced target specificity as compared to chemical pesticides and compatibility with integrated pest management (IPM) strategies.

Two-step separation strategy with mixed-mode chromatography to purify recombinant human serum albumin from Pichia pastoris culture broth

Mixed-mode chromatography (MMC) with the specially-designed ligands shows good adsorption selectivity for protein separation. In this study MMC was explored to purify recombinant human serum albumin (rHSA) from Pichia pastoris culture broth, which combined bind-elute and flow-through modes. Four mixed-mode resins were evaluated for their adsorption capacities of HSA. It was found that two home-made MMC resins showed special performances. IC-B-6FF resin with indole-3-acetic acid-cysteine ligand had high adsorption and selectivity towards HSA, and SFA-B-6FF resin with sulfapyridine ligand showed minimal binding to HSA. Therefore, a two-step MMC separation strategy was designed that IC-B-6FF was used to capture rHSA directly from yeast culture broth and then SFA-B-6FF resin was used to polish the remaining impurities by flow-through mode. The results revealed that the purity of rHSA reached 95.1% with a yield of 81.2% after the two-step separation. The removal rate of host cell proteins (HCPs) was 99.8% with final HCP level as low as 50 ppm. The results demonstrate that MMC combining bind-elute and flow-through modes has a promising potential to separate protein with high selectivity and process efficiency.

Hybrid Deep Modeling of a GS115 (Mut+) Pichia pastoris Culture with State–Space Reduction

Hybrid modeling workflows combining machine learning with mechanistic process descriptions are becoming essential tools for bioprocess digitalization. In this study, a hybrid deep modeling method with state–space reduction was developed and showcased with a P. pastoris GS115 Mut+ strain expressing a single-chain antibody fragment (scFv). Deep feedforward neural networks (FFNN) with varying depths were connected in series with bioreactor macroscopic material balance equations. The hybrid model structure was trained with a deep learning technique based on the adaptive moment estimation method (ADAM), semidirect sensitivity equations and stochastic regularization. A state–space reduction method was investigated based on a principal component analysis (PCA) of the cumulative reacted amount. Data of nine fed-batch P. pastoris 50 L cultivations served to validate the method. Hybrid deep models were developed describing process dynamics as a function of critical process parameters (CPPs). The state–space reduction method succeeded to decrease the hybrid model complexity by 60% and to improve the predictive power by 18.5% in relation to the nonreduced version. An exploratory design space analysis showed that the optimization of the feed of methanol and of inorganic elements has the potential to increase the scFv endpoint titer by 30% and 80%, respectively, in relation to the reference condition.

A Systematic Review of the Potential of Pichia pastoris (Komagataella phaffii) as an Alternative Host for Biologics Production.

The methylotrophic yeast Pichia pastoris is garnering interest as a chassis cell factory for the manufacture of recombinant proteins because it effectively satisfies the requirements of both laboratory and industrial set up. The optimisation of P. pastoris cultivation is still necessary due to strain- and product-specific problems such as promoter strength, methanol utilisation type, and culturing conditions to realize the high yields of heterologous protein(s) of interest. Techniques integrating genetic and process engineering have been used to overcome these problems. Insight into the Pichia as an expression system utilizing MUT pathway and the development of methanol free systems are highlighted in this systematic review. Recent developments in the improved production of proteins in P. pastoris by (i) diverse genetic engineering such as codon optimization and gene dosage; (ii) cultivating tactics including co-expression of chaperones; (iii) advances in the use of the 2A peptide system, and (iv) CRISPR/Cas technologies are widely discussed. We believe that by combining these strategies, P. pastoris will become a formidable platform for the production of high value therapeutic proteins.

Ultrathin membranes composed of branched polyethylenimine and poly[(o-cresyl glycidyl ether)-co-formaldehyde] for primary recovery of itaconic acid

Efficient primary recovery of building-block chemicals produced by microbial processes, such as biobased itaconic acid, is a key step for environmentally sustainable and economically feasible manufacturing processes. Ultrathin membranes have the potential to address these challenges. Here, ultrathin membranes composed of branched polyethylenimine and poly[(o-cresyl glycidyl ether)-co-formaldehyde] were fabricated using a facile spin coating procedure, and the composition of the casting solution was adjusted to improve the separation performance. The structural properties of the ultrathin membranes (intrinsic porosity, thickness, surface morphology, chemistry, charge, and wettability) were investigated and linked to the separation performance. By increasing the content of polyethylenimine in the casting solution, the transport rate of itaconic acid across the membranes increased up to 40 times. Membranes with a 4:1 mass ratio of polyethylenimine to poly[(o-cresyl glycidyl ether)-co-formaldehyde] reactive groups were selected, and their ability to recover itaconic acid from synthetic solutions and recombinant Pichia pastoris culture supernatant in diffusion-based mode was demonstrated. Experiments with pure solutions of supernatant components demonstrated that the membranes were equally selective for itaconic acid and potential cultivation byproducts and at least 7-times more selective for itaconic acid than culture medium components. Experiments with synthetic complex solutions and culture supernatant confirmed the high selectivity for itaconic acid: the separation performance was similar for complex and pure itaconic acid solutions, despite the significant difference in the complexity of these solutions. This study is the basis for a new technology to recover microbially produced building-block chemicals.

Prospects of biosimilar recombinant human growth hormone manufacture in the Republic of Moldova

In collaboration of SUMPh „N.Testemițanu”, the pharmaceutical company Balkan Pharmaceuticals and ICGEB the technology transfer during scale-up from the laboratory phase to the pilot phase of the processes of creating the working cell bank and growing Pichia pastoris strains in the Erlenmeyer flask were carried out. Pichia pastoris was selected for recombinant growth hormone as an expression system, which has many advantages over other organisms, including protein folding, post-translational changes in protein, and easy handling. The GH1 altered recombinant human somatropin gene was introduced into the genome of Pichia pastoris culture using restriction near the AOX1 gene promoter as a vector serving the pPIC9K plasmid. The GH1 gene was activated by the AOX1 gene promoter which can be easily induced by the presence of methanol in the culture medium. Subsequently, the „Prepro-alpha Factor Leader” sequence from Saccharomyces cerevisiae was added to the modified sequence so that the obtained somatropin could be secreted into the culture medium. In the technological transfer process the influence of the chosen technological processes on some parameters of the obtained product were examined, Technological Regulations were developed for short and long term storage processes of Pichia pastoris cells and for the process of inoculum preparation, inoculation and growth of P. pastoris strains with recombinant human growth hormone in the Erlenmeyer flask. Standard Operating Procedures were developed for the processes of creating the working cell bank, determining the in-vitro biological activity of the recombinant human growth hormone preparation, with subsequent implementation in the process of producing rhGH biosimilar at CS Balkan Pharmaceuticals Ltd.

Simultaneous enzyme production, Levan-type FOS synthesis and sugar by-products elimination using a recombinant Pichia pastoris strain expressing a levansucrase-endolevanase fusion enzyme

BackgroundAlthough Levan-type fructooligosaccharides (L-FOS) have been shown to exhibit prebiotic properties, no efficient methods for their large-scale production have been proposed. One alternative relies on the simultaneous levan synthesis from sucrose, followed by endolevanase hydrolysis. For this purpose, several options have been described, particularly through the synthesis of the corresponding enzymes in recombinant Escherichia coli. Major drawbacks still consist in the requirement of GRAS microorganisms for enzyme production, but mainly, the elimination of glucose and fructose, the reaction by-products.ResultsThe expression of a fusion enzyme between Bacillus licheniformis endolevanase (LevB1) and B. subtilis levansucrase (SacB) in Pichia pastoris cultures, coupled with the simultaneous synthesis of L-FOS from sucrose and the elimination of the residual monosaccharides, in a single one-pot process was developed. The proof of concept at 250 mL flask-level, resulted in 8.62 g of monosaccharide-free L-FOS and 12.83 gDCW of biomass, after 3 successive sucrose additions (30 g in total), that is a 28.7% yield (w L-FOS/w sucrose) over a period of 288 h.At a 1.5 L bioreactor-level, growth considerably increased and, after 59 h and two sucrose additions, 72.9 g of monosaccharide-free L-FOS and 22.77 gDCW of biomass were obtained from a total of 160 g of sucrose fed, corresponding to a 45.5% yield (w L-FOS/w sucrose), 1.6 higher than the flask system. The L-FOS obtained at flask-level had a DP lower than 20 fructose units, while at bioreactor-level smaller oligosaccharides were obtained, with a DP lower than 10, as a consequence of the lower endolevanase activity in the flask-level.ConclusionWe demonstrate here in a novel system, that P. pastoris cultures can simultaneously be used as comprehensive system to produce the enzyme and the enzymatic L-FOS synthesis with growth sustained by sucrose by-products. This system may be now the center of an optimization strategy for an efficient production of glucose and fructose free L-FOS, to make them available for their application as prebiotics. Besides, P. pastoris biomass also constitutes an interesting source of unicellular protein.

Effects of Temperature and pH on Recombinant Thaumatin II Production by Pichia pastoris

The sweet protein thaumatin is emerging as a promising sugar replacer in the market today, especially in the food and beverage sector. Rising demand for its production necessitates the large-scale extraction of this protein from its natural plant source, which can be limited in terms of raw material availability and production costs. Using a recombinant production technique via a yeast platform, specifically, Pichia pastoris, is more promising to achieve the product economically while maintaining batch-to-batch consistency. However, the bioproduction of recombinant proteins requires the identification of optimal process variables, constituting the maximal yield of the product of interest. These variables have a direct effect on the growth of the host organism and the secretion levels of the recombinant protein. In this study, two important environmental factors, pH, and temperature were assessed by cultivating P. pastoris in shake flasks to understand their influence on growth and the production levels of thaumatin II protein. The results from the pH study indicate that P. pastoris attained a higher viable cell density and secretion of protein at pH 6.0 compared to 5.0 when grown at 30 °C. Furthermore, within the three levels of temperatures investigated when grown at pH 6.0, the protein levels were the highest at 30 °C compared to 20 and 25 °C, whereas 25 °C exhibited the highest viable cell density. Interestingly, the trend observed from the qualitative effects of temperature and pH occurred in all the media that was investigated. These results broaden our understanding of how pH and temperature adjustment during P. pastoris cultivation aid in enhancing the production yields of thaumatin II prior to optimising the fed batch bioreactor operation.

HBsAg Production in Methanol Controlled <i>P. pastoris</i> GS115 Mut<sup>S</sup> Bioreactor Process

When producing recombinant proteins with Pichia pastoris, cultivation parameters, such as induction temperature, dissolved oxygen level and residual methanol concentration play a crucial role in product biosynthesis and subsequent purification, therefore to maximize protein yields, the optimization of these parameters is imperative. Two different Pichia pastoris cultivation strategies for HBsAg VLP production in a 5 L stirred-tank bioreactor and the influence of different cultivation parameters on product yield were investigated. Residual methanol concentrations were controlled at low (>0.01 g/L), medium (1.5-2.0 g/L) and high (5.0-6.0 g/L) levels using a PI-based feed rate control algorithm based on the online methanol sensor signal. Product was purified using a novel and rapid purification method including steps of ammonium sulfate precipitation, size-exclusion chromatography and hydrophobic interaction chromatography. Employing an in-situ methanol sensor probe, the PI-based methanol feed rate control algorithm provided residual methanol concentration control with an average deviation of ±0.4 g/L from set-point value. Employing a cultivation protocol with an increased methanol concentration controlled at 6.0 g/L and a reduced DO level below 10 %, resulting in a final dry cell biomass concentration of 140 g/L and purified HBsAg VLPs yield of 186 mg/L. Developed purification method proved advantageous to other described methods, as it did not include time consuming extraction and centrifugation steps.

An innovative strategy of recycling miscellaneous waste carbohydrates from high-fructose syrup production for Pichia pastoris fermentation

Recycling waste carbon source for fermentation is of great interests and high practical value. However, the process suffered some problems like unclear carbohydrate composition, low nutrition content, presence of various contaminants and high cost for pretreatment. Currently, the high-fructose syrup (HFS) production industry in China generates a large amount of waste liquid containing miscellaneous waste carbohydrates (MWC), which has a notable environmental impact. To recycle MWC for Pichia pastoris fermentation, the composition of MWC was analyzed and the fermentation processes were optimized. The composition of MWC was determined as 802.3 g L−1 total carbohydrate, 480 g L−1 glucose, 92 g L−1 fructose, 103.6 g L−1 maltose, 36.8 g L−1 maltotriose and 89.9 g L−1 other oligosaccharides with different degrees of polymerization (DPs 4–16). After the fermentation medium and conditions for pGAP/pAOX1-P. pastoris were optimized, MWC were employed as the alternative carbon source to culture the two strains to produce endo-β-1,3-glucanase in 7-L bioreactors. Using MWC for pGAP-P. pastoris culture, the dry cell weight (DCW) and endo-β-1,3-glucanase activity reached 69.1 g L−1 and 171.8 U mL−1, respectively, which were the same levels as in the batch using glucose as the carbon source. When using MWC and the self-designed DO-Stat-Time feeding strategy for pAOX1-P. pastoris culture, the maximum DCW and enzyme activity reached 83.6 g L−1 and 212.3 U mL−1, which improved 41.4% and 34.9%, respectively compared with the control group. This study established cleaner production procedures for both HFS and P. pastoris fermentation industries by considerably reducing environmental pollution and decreasing the production cost.

The role of propeptide-mediated autoinhibition and intermolecular chaperone in the maturation of cognate catalytic domain in leucine aminopeptidase

Leucyl aminopeptidase A from Aspergillus oryzae RIB40 (AO-LapA) is an exo-acting peptidase, widely utilised in food debittering applications. AO-LapA is secreted as a zymogen by the host and requires enzymatic cleavage of the autoinhibitory propeptide to reveal its full activity. Scarcity of structural data of zymogen aminopeptidases hampers a better understanding of the details of their molecular action of autoinhibition and how this might be utilised to improve the properties of such enzymes by recombinant methods for more effective bioprocessing. To address this gap in the literature, herein we report high-resolution crystal structures of recombinantly expressed AO-LapA precursor (AO-proLapA), mature LapA (AO-mLapA) and AO-mLapA complexed with reaction product l-leucine (AO-mLapA-Leu), all purified from Pichia pastoris culture supernatant. Our structures reveal a plausible molecular mechanism of LapA catalytic domain autoinhibition by propeptide and highlights the role of intramolecular chaperone (IMC). Our data suggest an absolute requirement for IMC in the maturation of cognate catalytic domain of AO-LapA. This observation is reinforced by our expression and refolding data of catalytic domain only (AO-refLapA) from Escherichia coli inclusion bodies, revealing a limited active conformation. Our work supports the notion that known synthetic aminopeptidase inhibitors and substrates mimic key polar contacts between propeptide and corresponding catalytic domain, demonstrated in our AO-proLapA zymogen crystal structure. Furthermore, understanding the atomic details of the autoinhibitory mechanism of cognate catalytic domains by native propeptides has wider reaching implications toward synthetic production of more effective inhibitors of bimetallic aminopeptidases and other dizinc enzymes that share an analogous reaction mechanism.

Capture and intermediate purification of human insulin precursor from Pichia pastoris culture using cation exchange chromatography

Purification has an important role in obtaining protein with a high degree of purity, particularly for human therapeutic purposes. Purification of pharmaceutical proteins requires several unit operations, involving chromatographic separation techniques. To increase purification efficiency and shorten process development, it is necessary to examine the chromatography system in performing a capture and intermediate purification in a single step. We use one of the best Pichia pastoris clones obtained from previous studies to produce a human insulin precursor (HIP). To capture and purify HIP from the culture, we clarified the cells through centrifugation and filtration. The supernatant was then loaded into a cation exchange column. Purification was carried on by two-step elution and monitored based on UV absorbance. Effects of loading concentration, flow rate, and pH of samples were evaluated. Fractions of elution were collected and verified by SDS-PAGE. Concentrations of HIP protein were quantified by using ImageJ by incorporating lysozyme as standard and reversed phase HPLC. Loading concentration and pH of the sample have an impact on the recovery. In this study, the best HIP recovery at ∼ 47% resulted from purification with 10% volume of loading concentration and 500 ml loading volume.

Systematic review and meta-analysis for the biotechnological production of THC in Morocco

Cannabinoids have promising therapeutic potential. Δ9-tetrahydrocannabinol (THC), the most important psychotropic active ingredient of Cannabis Sativa L, has been the subject of several chemical, pharmacological and biosynthetic studies. In this context, a meta-analysis of biotechnological processes applied to the production of recombinant cannabinoid THC worldwide was carried out. The objective was to highlight the potential of these processes on the Moroccan variety of Cannabis Sativa. The PubMed, ScienceDirect and Web of Science search motors were used to search for original scientific work presenting biotechnological tools used in the production of THC. The scientific articles exploited are those published before the end of 2020. Succinct analysis of the experimental work performed showed that expression of the gene encoding Cannabis Sativa L. THCA synthase was performed on prokaryotic and eukaryotic expression systems. Currently, no functional expression could be obtained in E. coli. Whereas, production of recombinant protein (THCA Synthase) associated with significant enzymatic activity was obtained in P. pastoris cultures (F. Saccharomycetaceae). The exploitation of the sequences showed the presence of a large similarity between the THCA Synthase gene of cannabis Sativa of the Moroccan variety and the mRNA precursor of the same gene reported in several studies. This will allow us to use a specific signal sequence of choice for an adopted expression host, in order to produce the recombinant THCA synthase enzyme from the Moroccan Cannabis Sativa L strain on P. pastoris cultures.

Biocalorimetric monitoring of glycoengineered P. pastoris cultivation for the production of recombinant huIFNα2b: A quantitative study based on mixed feeding strategies.

Real-time monitoring of glycoengineered Pichia pastoris by employing process analytical technology (PAT) tools is vital for gaining deeper insights into the therapeutic protein production process. The present study focuses on influence of mixed feed carbon substrates during the induction phases of glycoengineered P. pastoris cultivation, for recombinant human interferon α2b (huIFNα2b) production by employing calorimetric (biological heat rate, q B ) and respirometric (oxygen uptake rate and carbon dioxide evolution rate) measurements. Mixed feed stream of carbon substrates (methanol + glycerol, methanol + sorbitol) at a predetermined "C-molar ratios" were added during the induction phases. Methanol- and sorbitol-based mixed feeding approach resulted in an improved huIFNα2b titer of 288 mg/L by channeling of methanol predominantly towards an optimal functioning of AOX expression system. A stand-off between biomass yield coefficient, degree of reduction of methanol and its cosubstrate (glycerol and sorbitol) determines the fraction of carbon energy channeled toward biomass and protein production, under strict aerobic conditions. Calorespirometric monitoring and assessment of thermal yields enables a reliable prediction of process variables, leading to futuristic efficient PAT-based feed rate control.

Expression and detergent free purification and reconstitution of the plant plasma membrane Na+/H+ antiporter SOS1 overexpressed in Pichia pastoris

The plant plasma membrane Na+/H+ antiporter SOS1 (Salt Overlay Sensitive 1) of Arabidopsis thaliana is the major transporter extruding Na+ out of cells in exchange for an intracellular H+. The sodium extrusion process maintains a low intracellular Na+ concentration and thereby facilitates salt tolerance. A. thaliana SOS1 consists of 1146 amino acids, with the first 450 in a N-terminal membrane transport domain and the balance forming a cytosolic regulatory domain. For studies on characterization of the protein, two different constructs of SOS1 comprising of the residues 28 to 460 and 28 to 990 were cloned and overexpressed in methylotropic yeast strain of Pichia pastoris with a C-terminal histidine tag using the expression vector pPICZA. Styrene malic acid copolymers (SMA) were used as a cost-effective alternative to detergent for solubilization and isolation of this membrane protein. Immobilized Ni2+-ion affinity chromatography was used to purify the expressed protein resulting in a yield of ~0.6–2 mg of SOS1 per liter of Pichia pastoris culture. The SMA purified protein containing amino acids 28 to 990 was directly reconstituted into liposomes for determination of Na+ transport activity and was functionally active. However, similar reconstitution with amino acids 28–460 did not yield a functional protein. Other results have shown that the truncated SOS1 protein at amino acid 481 is active, which infers the presence of an element between residues 461–481 which is necessary for SOS1 activity. This region contains several conserved segments that may be important in SOS1 structure and function.

Purification of Recombinant Glycoproteins from Pichia pastoris Culture Supernatants.

Pichia pastoris is a common host organism for the production of recombinant proteins. While unglycosylated recombinant proteins derived from this yeast can be purified efficiently by only a few conventional chromatography steps, the purification of glycosylated recombinant proteins is a very challenging process due to the intrinsic feature of the yeast of hypermannosylation. The resulting vast glycosylation pattern on the recombinant target protein masks its physicochemical properties hampering a conventional downstream process. Here, we describe a fast and efficient two-step chromatography strategy, where both steps are operated in flow-through mode, to purify recombinant glycoproteins from P. pastoris culture supernatants.

Fluorescence-based real time monitoring and diagnostics of recombinant Pichia pastoris cultivations in a bioreactor.

This study describes the application of the multivariate curve resolution (MCR) analysis technique for real-time analysis of culture fluorescence during recombinant Pichia pastoris cultivation in a bioreactor. Fluorescence spectra were acquired with an on-line dual excitation wavelength fluorometer and then used to develop a real time MCR-based bioprocess monitoring and diagnostics tool. Initial bioreactor experiments using two similar recombinant antibody secreting P. pastoris cell lines showed significant differences in protein production. To distinguish between the contributions of operating conditions and the specific cell line's genetic composition to the observed differences in protein production, the bioreactor experiments were repeated and accompanied by real time MCR analysis. The tests demonstrated high sensitivity of MCR-derived "pure concentration" profiles to growth as well as to initial conditions, thus enabling real-time cultivation process trend diagnostics and fault detection. © 2018 Her Majesty the Queen in Right of Canada © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2761, 2019.

In situ removal of consensus dengue virus envelope protein domain III fused to hydrophobin in Pichia pastoris cultures

This work describes a novel strategy for the integrated expression and purification of recombinant proteins in Pichia pastoris cultures. Hydrophobins can be used as fusion tags, proteins fused to them alter their hydrophobicity and can be purified by aqueous two-phase systems (ATPS) based on non-ionic surfactants. Here, the consensus dengue virus envelope protein domain III fused to hydrophobin I of Trichoderma reesei was expressed in Pichia pastoris cultures and an in situ product removal by an ATPS using a non-ionic detergent, (Triton X-114) was performed. The protein was produced and purified directly from the yeast culture supernatant both efficiently and with no loss. The purified protein was properly immobilized by adsorption in solid phase and recognized by anti-dengue antibodies, showing its potential for the development of an indirect immunoassay for dengue virus.

Expression, activation and processing of a novel plant milk-clotting aspartic protease in Pichia pastoris

Galium verum, also known as Lady’s Bedstraw or Cheese Rennet, is an herbaceous perennial plant traditionally used in cheese-making. We used RACE PCR to isolate novel enzymes from Galium verum with the ability to clot milk. This approach generated two cDNA sequences (named preprogaline A and B) encoding proteins displaying the typical plant aspartic protease primary structure. Preprogaline B was expressed in the yeast Pichia pastoris, after deleting and replacing its original signal peptide with the yeast α-factor signal peptide from Saccharomyces cerevisiae. The secreted recombinant protein was obtained by growing P. pastoris in YPD medium and had the ability to clot milk. The mature form of progaline B is a heterodimeric glycosylated enzyme, with a molecular weight of approximately 48 kDa, that contains a heavy (30.7 kDa) and a light (13.5 kDa) polypeptide chains linked by disulfide bonds. Western blot analysis revealed that progaline B is activated by the acidification of the yeast culture medium and that enzymatic activation requires two steps. First the precursor protein is cleaved into two polypeptide chains by partial removal of the plant-specific insert (PSI) present in plant aspartic proteases; this is later followed by propeptide removal. By altering the pH of the P. pastoris culture medium, we were able to obtain either active or inactive forms of the enzyme. Recombinant progaline B displayed a κ-casein hydrolysis pattern analogous to those produced by the animal and microbial coagulants currently used in the dairy industry, but it exhibited a different digestion profile on α- and β-caseins. The plant protease progaline B displays milk-clotting activities suitable for the production of novel dairy products.

High-throughput screening and optimization of mixed-mode resins for human serum albumin separation with microtiter filter plate

High-throughput approaches become increasingly important on process development for protein separation. In this work, four mixed-mode resins (Capto adhere, Capto MMC, MX-Trp–650 M and Nuvia cPrime) were investigated for human serum albumin (HSA) separation and compared with two ion-exchangers (Q Sepharose FF and SP Sepharose FF). High-throughput method based on microtiter filter plate was used to determine adsorption properties and optimize separation conditions. For resins with anion-exchange groups alkaline condition would be favorable for HSA adsorption, while acidic condition for resins with cation-exchange groups. Mixed-mode resins showed obviously salt-tolerant properties. Salt addition had little effect on Nuvia cPrime, and could promote HSA adsorption for Capto MMC and MX-Trp–650 M. The elution conditions were also optimized and high recovery could be obtained. In addition, the optimized conditions obtained by high-throughput approaches were verified by lab-scale column chromatography. It was found that Capto MMC and MX-Trp–650 M would be more suitable for HSA separation and DBC10% could reach more than 40 mg/ml under 200 mM NaCl addition. Finally, rHSA separation from P. pastoris culture broth was tested with MX-Trp–650 M, and high purity and recovery could be obtained. This study demonstrated that high-throughput screening method is a powerful tool to optimize the chromatographic separation. Mixed-mode cation-exchange resins showed an efficient practicability for HSA separation with high capacity, suitable salt-tolerance and high elution recovery.