Improving AOX1 promoter efficiency by overexpression of Mit1 transcription factor.

BACKGROUND: Reprogramming in transcriptional regulation provides an effective tool for adjusting cellular metabolic activities. The strong methanol-inducible alcohol oxidase-1 promoter (pAOX1) is commonly used for heterologous gene expression in the yeast Pichia pastoris. Here, we present a novel Pichia pastoris strain engineered to co-express methanol-induced transcription factor 1 (Mit1) and the target protein. Mit1 upregulates pAOX1 in response to methanol. METHODS AND RESULTS: Two model proteins (VEGF and eGFP) have been used as the target proteins under the control of pAOX1. The sequence of Mit1 had obtained from the yeast genome and likewise cloned under the control of pAOX1. The results indicated a 1.9 and 2.2 fold increase in the detected VEGF and eGFP, respectively, when co-expressed with Mit1. Furthermore, the double-recombinant cells, containing Mit-1 and eGFP, produced 1.3 fold more eGFP when the methanol feeding concentration was doubled. The real-time PCR indicated a slight increase in the Mit1 expression, probably due to the negative regulatory feedback loop that exists for the intrinsic yeast Mit1. Overexpression of Mit1 also led to duplication of AOX1 enzyme activity, which may enhance the yeast cells' capacity for methanol detoxification. CONCLUSION: Overexpression of Mit1 could be considered a promising strategy for upregulation of target recombinant proteins in Pichia pastoris. Intracellular overexpression of Mit1 upregulates the heterologous target gene (eGFP) production, which is expressed under the control of pAOX1.

Evaluation of Erythroferrone, Hepcidin, and Iron Overload Status in Iraqi Transfusion-Dependent ß-Thalassemia Major Patients.

Patients with ß-thalassemia major (ß-TM) show ineffective erythropoiesis and iron overload, which is the leading cause of mortality and organ injury. The present study aimed to investigate the relationships between two iron regulatory hormones, hepcidin and erythroferrone (ERFE) levels, and iron status parameters in Iraqi patients with ß-TM. Iron status parameters and hormones were measured in 60 patients and compared with 30 healthy controls. The results indicated significant changes in different iron status parameters, while ferritin with the ∼11-fold increase showed the most change. Significant reduction in hepcidin and an increase in ERFE levels were detected in patients when compared to the control group, while no direct correlation was identified with the other measured iron status parameters. The receiver operating characteristic (ROC) analysis showed that the z-score of the composite of ERFE + ferritin has a full diagnostic ability for ß-TM. In conclusion, our findings indicated the correlation between different iron status parameters and ferritin as the leading predictor of iron overload and two main iron regulatory hormones.

Promoting hepatogenic differentiation of human mesenchymal stem cells using a novel laminin-containing gelatin cryogel scaffold.

Liver transplantation is the only definitive treatment currently available for acute and chronic liver failure. However, this approach has been restricted by complications including rejection and infection. Tissue engineering approaches using stem cell-derived functional hepatic cells offer a potential alternative. Using biologically compatible scaffolds is an important complementary key to achieve optimal construct for hepatic replacement. In the present study, to optimize the differentiation of human adipose-derived mesenchymal stem cells (ADMSCs) toward hepatocyte-like cells, a previously described gelatin cryogel was optimized and improved by laminin, the major component of basal lamina. The ADMSCs seeded on the scaffold displayed increased attachment in the presence of laminin and the MTT assay showed good compatibility for cell proliferation. The differentiation of stem cells were evaluated using glycogen staining, urea secretion measurement, hepatocyte specific cell surface analysis and gene expression analysis. The results of tests indicated that laminin protein and gelatin cryogel 3D scaffold, each on its own, enhanced hepatogenic differentiation of ADMSCs. However, when laminin immobilized on the gelatin cryogel surface, the differentiation was promoted significantly and the resulting cells showed striking similarity to HepG2 in terms of expressing studied hepatocyte markers.

Changes in biophysical characteristics of PFN1 due to mutation causing amyotrophic lateral sclerosis.

Single amino acid mutations in profilin 1 (PFN1) have been found to cause amyotrophic lateral sclerosis (ALS). Recently, we developed a mouse model for ALS using a PFN1 mutation (glycine 118 to valine, G118V), and we are now interested in understanding how PFN1 becomes toxically lethal with only one amino acid substitution. Therefore, we studied mutation-related changes in the PFN1 protein and hypothesized that such changes significantly disturb its structure. Initially, we expressed and studied the purified PFN1WT and PFN1G118V proteins from bacterial culture. We found that the PFN1G118V protein has a different mean residue ellipticity, as measured by far-UV circular dichroism, accompanied by a spectral shift. The intrinsic fluorescence of PFN1G118V showed a small fluctuation in maximum fluorescence absorption and intensity. Moreover, we examined the time course of PFN1 aggregation using SDS-PAGE, western blotting, and MALDI-TOF/TOF and found that compared with PFN1WT, PFN1G118V had an increased tendency to form aggregates. Dynamic light scattering data confirmed this, showing a larger size distribution for PFN1G118V. Our data explain why PFN1G118V tends to aggregate, a phenotype that may be the basis for its neurotoxicity.

The enhancement of differentiating adipose derived mesenchymal stem cells toward hepatocyte like cells using gelatin cryogel scaffold.

Liver tissue engineering creates a promising methodology for developing functional tissue to restore or improve the function of lost or damaged liver by using appropriate cells and biologically compatible scaffolds. The present paper aims to study the hepatogenic potential of human adipose derived mesenchymal stem cells (hADSCs) on a 3D gelatin scaffold in vitro. For this purpose, mesenchymal stem cells were isolated from human adipose tissue and characterized by flowcytometry analysis and mesodermal lineage differentiation capacity. Then, porous cryogel scaffolds were fabricated by cryogelating the gelatin using glutaraldehyde as the crosslinking agent. The structure of the scaffolds as well as the adhesion and proliferation of the cells were then determined by Scanning Electron Microscopy (SEM) analysis and MTT assay, respectively. The efficiency of hepatic differentiation of hADSCs on 2D and 3D culture systems has been assessed by means of morphological, cytological, molecular and biochemical approaches. Based on the results of flowcytometry, the isolated cells were positive for hMSC specific markers and negative for hematopoietic markers. Further, the multipotency of these cells was confirmed by adipogenic and osteogenic differentiation and the highly porous structure of scaffolds was characterized by SEM images. Biocompatibility was observed in the fabricated gelatin scaffolds and the adhesion and proliferation of hADSCs were promoted without any cytotoxicity effects. In addition, compared to 2D TCPS, the fabricated scaffolds provided more appropriate microenvironment resulting in promoting the differentiation of hADSCs toward hepatocyte-like cells with higher expression of hepatocyte-specific markers and appropriate functional characteristics such as increased levels of urea biosynthesis and glycogen storage. Finally, the created 3D gelatin scaffold could provide an appropriate matrix for hepatogenic differentiation of hADSCs, which could be considered for liver tissue engineering applications.

Promoters in Pichia pastoris: A Toolbox for Fine-Tuned Gene Expression.

This chapter reviews the different promoters used to control gene expression in the yeast Pichia pastoris, mainly for recombinant protein production. It covers natural inducible, derepressed, and constitutive promoters, as well as engineered synthetic/hybrid promoters, orthologous promoters from related yeasts, and emerging bidirectional promoters. Key examples, characteristics, and regulatory mechanisms are discussed for each promoter class. Recent efforts in promoter engineering through rational design, mutagenesis, and computational approaches are also highlighted. Looking ahead, we anticipate further developments that will enhance promoter design for Pichia pastoris. Overall, this comprehensive overview underscores the importance of promoter choice and engineering for fully harnessing Pichia pastoris biotechnological potential.

Green synthesis of yeast cell wall-derived carbon quantum dots with multiple biological activities.

Hypothesis: Yeast cell walls are a sustainable biomass source containing carbon and other elements like phosphorus. Converting cell walls into valuable nanomaterials like carbon quantum dots (CQDs) is of interest. Experiments: Cell walls from Saccharomyces cerevisiae were hydrothermally treated in 0.5 M H2SO4 to produce CQDs. Multiple analytical techniques were utilized to confirm phosphorus-doping (P-CQDs), characterize the fluorescence properties, determine quantum yield, and evaluate the sensing, antimicrobial, photocatalytic, and antioxidant capacities. Findings: A successful synthesis of P-CQDs was achieved with strong blue fluorescence under UV excitation, 19 % quantum yield, and excellent stability. The P-CQDs showed sensitive fluorescence quenching in response to ferric ions with a 201 nM detection limit. Antibacterial effects against Escherichia coli and Staphylococcus aureus were demonstrated. P-CQDs also exhibited dye degradation under sunlight and antioxidant activity. So, the prepared P-CQDs displayed promising multifunctional capabilities for metal ion detection, disinfection, and environmental remediation. Further research is required to fully realize and implement the multifunctional potential of P-CQDs in real-world applications.

Electrospun nanofibrous wound dressings with enhanced efficiency through carbon quantum dots and citrate incorporation.

Nanofibers show promise for wound healing by facilitating active agent delivery, moisture retention, and tissue regeneration. However, selecting suitable dressings for diverse wound types and managing varying exudate levels remains challenging. This study synthesized carbon quantum dots (CQDs) from citrate salt and thiourea using a hydrothermal method. The CQDs displayed antibacterial activity against Staphylococcus aureus and Escherichia coli. A nanoscaffold comprising gelatin, chitosan, and polycaprolactone (GCP) was synthesized and enhanced with silver nanoparticle-coated CQDs (Ag-CQDs) to form GCP-Q, while citrate addition yielded GCP-QC. Multiple analytical techniques, including electron microscopy, FT-IR spectroscopy, dynamic light scattering, UV-Vis, photoluminescence, X-ray diffraction, porosity, degradability, contact angle, and histopathology assessments characterized the CQDs and nanofibers. Integration of CQDs and citrate into the GCP nanofibers increased porosity, hydrophilicity, and degradability-properties favorable for wound healing. Hematoxylin and eosin staining showed accelerated wound closure with GCP-Q and GCP-QC compared to GCP alone. Overall, GCP-Q and GCP-QC nanofibers exhibit significant potential for skin tissue engineering applications.

Hansenula polymorpha methanol metabolism genes enhance recombinant protein production in Komagataella phaffi.

Recombinant protein production in Komagataella phaffi (K. phaffi), a widely utilized host organism, can be optimized by enhancing the metabolic flux in the central carbon metabolism pathways. The methanol utilization pathway (MUT) during methanol-based growth plays a crucial role in providing precursors and energy for cell growth and development. This study investigated the impact of boosting the methanol dissimilation pathway, a branch of MUT that plays a vital role in detoxifying formaldehyde and providing energy in the form of NADH, in K. phaffi. This was achieved by integrating two orthologous genes from Hansenula polymorpha into the K. phaffi genome: formaldehyde dehydrogenase (HpFLD) and formate dehydrogenase (HpFMDH). The HpFLD and HpFMDH genes were isolated from the Hansenula polymorpha genome and inserted under the regulation of the pAOX1 promoter in the genome of recombinant K. phaffi that already contained a single copy of model protein genes (eGFP or EGII). The expression levels of these model proteins were assessed through protein activity assays and gene expression analysis. The findings revealed that while both orthologous genes positively influenced model protein production, HpFMDH exhibited a more pronounced upregulation in expression compared to HpFLD. Co-expression of both orthologous genes demonstrated synergistic effects, resulting in approximately a twofold increase in the levels of the model proteins detected. This study provides valuable insights into enhancing the production capacity of recombinant proteins in K. phaffi.

Effectiveness of human adipose tissue-derived mesenchymal stem cells expressing alpha-1 antitrypsin gene in liver fibrosis: a study in mice.

Aim: The present study examined the protective potential of human adipose tissue-derived mesenchymal stem cells (hASCs) modified to overexpress alpha-1 antitrypsin (AAT), in a mouse model of the liver fibrosis. Background: For the treatment of end-stage liver diseases, cell therapy has emerged as a promising noninvasive alternative to liver transplantation. Mesenchymal stem cells (MSCs) are being evaluated due to their dual capabilities of promoting liver regeneration and modulating the pathogenic inflammation of the immune system. Methods: Liver fibrosis was induced in mice via the intraperitoneal injection of carbon tetrachloride (CCl4). MSCs were extracted from the human adipose tissue. After stemness confirmation, the cells were transduced with the lentiviruses containing the AAT gene, and then injected into the mice's tail vein. Fourteen days' post-transplantation, mice were sacrificed, and blood and tissue samples were collected for analysis. Important liver enzymes, including alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), albumin, and total bilirubin (TB), were measured. Histological studies were carried out using the hematoxylin and eosin (H&E), as well as Masson's trichrome (MT) staining. Results: Compared to hASCs, treatment with AAT-hASCs resulted in greater reductions in ALT, AST, ALP, and TB, as well as normalized albumin levels. AAT-hASCs promoted enhanced liver regeneration histologically, likely attributable to anti-inflammatory and anti-proteolytic properties of AAT. Conclusion: These findings indicate AAT-engineered hASCs as a promising cell-gene therapy candidate for further study in liver cirrhosis models.

Simulation and experimental study of a cold atmospheric pressure plasma and comparison of efficiency in boosting recombinant Endoglucanase II production in Pichia pastoris.

Recombinant proteins are essential in various industries, and scientists employ genetic engineering and synthetic biology to enhance the host cell's protein production capacity. Stress response pathways have been found effective in augmenting protein secretion. Cold atmospheric pressure plasma (CAP) can induce oxidative stress and enhance protein production. Previous studies have confirmed the applicability of CAP jets on Phytase and green fluorescent protein (GFP) production in Pichia pastoris hosts. This study investigates the effect of CAP treatment on another valuable recombinant protein, Endoglucanase II (EgII), integrated into the Pichia pastoris genome. The results demonstrated that plasma induction via two different ignition modes: sinusoidal alternating current (AC) and pulsed direct current (DC) for 120, 180, and 240 s has boosted protein secretion without affecting cell growth and viability. The AC-driven jet exhibited a higher percentage increase in secretion, up to 45%. Simulation of plasma function using COMSOL software provided a pattern of electron temperature (Te) and density distribution, which determine the plasma cocktail's chemistry and reactive species production. Furthermore, electron density (ne) and temperature were estimated from the recorded optical spectrum. The difference in electron properties may explain the moderately different impressions on expression capability. However, cell engineering to improve secretion often remains a trial-and-error approach, and improvements are, at least partially, specific to the protein produced.

Cold atmospheric plasma treatment enhances recombinant model protein production in yeast Pichia pastoris.

Cold atmospheric pressure plasma (CAP) has been described as a novel technology with expanding applications in biomedicine and biotechnology. In the present study, we provide a mildly stressful condition using non-lethal doses of CAP (120, 180, and 240 s) and evaluate its potential benefits on the recombinant production of a model protein (enhanced green fluorescent protein (eGFP)) in yeast Pichia pastoris. The measured eGFP fluorescence augmented proportional to CAP exposure time. After 240 s treatment with CAP, the measured fluorescent intensity of culture supernatant (after 72 h) and results of real-time PCR (after 24 h) indicated an 84% and 76% increase in activity and related RNA concentration, respectively. Real-time analysis of a list of genes involved in oxidative stress response revealed a significant and durable improvement in their expression at five h and 24 h following CAP exposure. The improvement of the recombinant model protein production may be partly explained by the impact of the RONS on cellular constituents and altering the expression of specific stress genes. In conclusion, using CAP strategy may be considered a valuable strategy to improve recombinant protein production, and deciphering the molecular background mechanism could be inspiring in the reverse metabolic engineering of host cells.

An overview of the vaccine platforms to combat COVID-19 with a focus on the subunit vaccines.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging virus that has caused the recent coronavirus disease (COVID-19) global pandemic. The current approved COVID-19 vaccines have shown considerable efficiency against hospitalization and death. However, the continuation of the pandemic for more than two years and the likelihood of new strain emergence despite the global rollout of vaccination highlight the immediate need for the development and improvement of vaccines. mRNA, viral vector, and inactivated virus vaccine platforms were the first members of the worldwide approved vaccine list. Subunit vaccines. which are vaccines based on synthetic peptides or recombinant proteins, have been used in lower numbers and limited countries. The unavoidable advantages of this platform, including safety and precise immune targeting, make it a promising vaccine with wider global use in the near future. This review article summarizes the current knowledge on different vaccine platforms, focusing on the subunit vaccines and their clinical trial advancements against COVID-19.

Expression, characterization, and activity optimization of a novel cellulase from the thermophilic bacteria Cohnella sp. A01.

Cellulases are hydrolytic enzymes with wide scientific and industrial applications. We described a novel cellulase, CelC307, from the thermophilic indigenous Cohnella sp. A01. The 3-D structure of the CelC307 was predicted by comparative modeling. Docking of CelC307 with specific inhibitors and molecular dynamic (MD) simulation revealed that these ligands bound in a non-competitive manner. The CelC307 protein was purified and characterized after recombinant expression in Escherichia coli (E. coli) BL21. Using CMC 1% as the substrate, the thermodynamic values were determined as Km 0.46 mM, kcat 104.30 × 10-3 (S-1), and kcat/Km 226.73 (M-1 S-1). The CelC307 was optimally active at 40 °C and pH 7.0. The culture condition was optimized for improved CelC307 expression using Plackett-Burman and Box-Behnken design as follows: temperature 20 °C, pH 7.5, and inoculation concentration with an OD600 = 1. The endoglucanase activity was positively modulated in the presence of Na+, Li+, Ca2+, 2-mercaptoethanol (2-ME), and glycerol. The thermodynamic parameters calculated for CelC307 confirmed its inherent thermostability. The characterized CelC307 may be a suitable candidate for various biotechnological applications.

Expression of functional eGFP-fused antigen-binding fragment of ranibizumab in Pichia pastoris.

Introduction: Ranibizumab is a mouse monoclonal antibody fragment antigen-binding (Fab) against human vascular endothelial growth factor-A (VEGF-A), inhibiting angiogenesis. This antibody is commercially produced in Escherichia coli host and used to treat wet age-related macular degeneration (AMD). Methods: In this study, the heavy and light chains of ranibizumab were expressed in Pichia pastoris. The expressed chains were incubated overnight at 4°C for interaction. The formation of an active structure was evaluated based on the interaction with substrate VEGF-A using an indirect ELISA, and an electrochemical setup. Furthermore, reconstruction of split enhanced green fluorescent protein (eGFP) reporter, chimerized at the C-terminus of the heavy and light chains, was used to characterize chains' interaction. Results: P. pastoris efficiently expressed designed constructs and secreted them into the culture medium. The anti-Fab antibody detected the constructed Fab structure in western blot analysis. Reconstruction of the split reporter confirmed the interaction between heavy and light chains. The designed ELISA and electrochemical setup results verified the binding activity of the recombinant Fab structure against VEGF-A. Conclusion: In this work, we indicated that the heavy and light chains of ranibizumab Fab fragments (with or without linkage to split parts of eGFP protein) were produced in P. pastoris. The fluorescence of reconstructed eGFP was detected after incubating the equal ratio of chimeric-heavy and light chains. Immunoassay and electrochemical tests verified the bioactivity of constructed Fab. The data suggested that P. pastoris could be considered a potential efficient eukaryotic host for ranibizumab production.

A highly-sensitive vascular endothelial growth factor-A(165) immunosensor, as a tool for early detection of cancer.

Biomarkers can be ideal indicators for assessing the risk of the presence of a disease. In this study, a label-free electrochemical biosensor was designed to quantify the vascular endothelial growth factor A (165) (VEGF-A(165)) antigen, using reduced graphene oxide-gold nanoparticle for early detection of breast cancer. The conductivity of gold nanoparticle along with its biocompatibility provide an enhanced surface, suitable for anti-VEGF antibody immobilization. 11-mercaptoundecanoic acid was used to facilitate a single-step and convenient bonding of the antibodies to the surface, compared to previous studies. The dynamic range of the biosensor was between 20 to 120 pg/ml and its limit of detection of the biomarker VEGF-A(165) was obtained to be about 0.007 pg/ml, using different electric signal transduction modes. Hence, the biosensor is a beneficial immunosensor with high sensitivity and ideal dynamic range for early-stage diagnosis of breast cancer and other cancers diseases associated with expression of VEGF-A(165). The as-prepared immunosensor could be efficiently employed for designing a point-of-care diagnostic platform.

A novel thermostable alkaline histamine oxidase from Glutamicibacter sp. N1A3101, induced by histamine and its analogue betahistine.

Biogenic amines (BAs) are low molecular weight organic bases formed by natural amino acids decarboxylation and trigger an array of toxicological effects in humans and animals. Bacterial amine oxidases enzymes are determined as practical tools to implement the rapid quantification of BAs in foods. Our study set out to obtain a new efficient, amine oxidase enzyme for developing new enzyme-based quantification of histamine. The soils from different sources were screened using histamine as sole carbon and nitrogen sources, and histamine oxidase producing bacteria were selected and identified using specific primers for histamine oxidase (HOD) gene. The HOD gene of six strains, out of 26 isolated histamine-utilizing bacteria, were amplified using our designed primers. The HOD enzyme from Glutamicibacter sp. N1A3101, isolated from nettle soil, was found to be thermostable and showed the highest substrate specificity toward the histamine and with no detected activity in the presence of putrescine, cadaverine, spermine, and spermidine. Its oxidation activity toward tyramine was lower than other HOD reported so far. The isolated enzyme was stable at 60 °C for 30 min and showed pH stability ranging from 6 to 9. Furthermore, we indicated the induction of identified HOD activity in the presence of betahistine as well, with nearly equal efficiency and without the consumption of the substrate.

pMOX: a new powerful promoter for recombinant protein production in yeast Pichia pastoris.

AOX1 promoter (pAOX1) is a robust inducible promoter highly preferred for the production of recombinant proteins in Pichia pastoris (P. pastoris). However, repression by other carbon sources and induction by methanol, which is a fire hazard chemical and undesirable for industrial production, are remarkable drawbacks in large-scale use of this promoter. Hence, novel strong regulatory promoters are highly desired. In the present study, the promoter region of methanol oxidase gene (pMOX), from Hansenula polymorpha, was explored for the heterologous expression of foreign proteins in protease deficient and wild type P. pastoris strains. The promoter region of MOX was isolated and replaced with the pAOX1 in the pPINK-HC plasmid. The activity of pMOX and pAOX1 was compared using endoglucanase 3 (CMC3) and endoglucanase II (EgII) enzymes as the reporter proteins. Evaluation of carbon sources on pMOX activity showed complete inactivation in the presence of xylose and sorbitol and high activity by glycerol, glucose and methanol feeding. Furthermore, the results indicated that increasing the gene dosage and using protease deficient-trait significantly increased CMC3 and EgII expression under the control of pMOX. In conclusion, in this study, a new small powerful and methanol-free promoter is introduced for recombinant protein production in yeast P. pastoris.

Structural and biochemical characterization of a novel thermophilic Coh01147 protease.

Proteases play an essential role in living organisms and represent one of the largest groups of industrial enzymes. The aim of this work was recombinant production and characterization of a newly identified thermostable protease 1147 from thermophilum indigenous Cohnella sp. A01. Phylogenetic tree analysis showed that protease 1147 is closely related to the cysteine proteases from DJ-1/ThiJ/PfpI superfamily, with the conserved catalytic tetrad. Structural prediction using MODELLER 9v7 indicated that protease 1147 has an overall α/ß sandwich tertiary structure. The gene of protease 1147 was cloned and expressed in Escherichia coli (E. coli) BL21. The recombinant protease 1147 appeared as a homogenous band of 18 kDa in SDS-PAGE, which was verified by western blot and zymography. The recombinant protein was purified with a yield of approximately 88% in a single step using Ni-NTA affinity chromatography. Furthermore, a rapid one-step thermal shock procedure was successfully implemented to purify the protein with a yield of 73%. Using casein as the substrate, Km, and kcat, kcat/Km values of 13.72 mM, 3.143 × 10-3 (s-1), and 0.381 (M-1 S-1) were obtained, respectively. The maximum protease activity was detected at pH = 7 and 60°C with the inactivation rate constant (kin) of 2.10 × 10-3 (m-1), and half-life (t1/2) of 330.07 min. Protease 1147 exhibited excellent stability to organic solvent, metal ions, and 1% SDS. The protease activity was significantly enhanced by Tween 20 and Tween 80 and suppressed by cysteine protease specific inhibitors. Docking results and molecular dynamics (MD) simulation revealed that Tween 20 interacted with protease 1147 via hydrogen bonds and made the structure more stable. CD and fluorescence spectra indicated structural changes taking place at 100°C, very basic and acidic pH, and in the presence of Tween 20. These properties make this newly characterized protease a potential candidate for various biotechnological applications.

Enhancing chimeric hydrophobin II-vascular endothelial growth factor A165 expression in Pichia pastoris and its efficient purification using hydrophobin counterpart.

We report cloning and expressing of recombinant human VEGF-A165, fused at the N-terminal with Hydrophobin II (HFBII) from Trichoderma reseei, in yeast Pichia pastoris. We validated the construct using SDS-PAGE and ELISA against VEGF-A165 and efficiently performed protein purification and enrichment based on HFBII counterpart and using an aqueous two-phase system (ATPS) with nonionic surfactant X-114. We studied the effects of various culture medium additives and interaction effects of positive factors to increase the recombinant HFBII-VEGF-A165 production. Supplementing the Pichia pastoris cell culture medium with Mg2+, Polysorbate 20 (PS 20), and 4-phenylbutyrate (PBA) improved the expression of the chimeric protein. Orthogonal experiments showed that the optimal condition to achieve maximal HFBII-VEGF-A165 production was with the addition of PBA, PS 20, and MgSO4. Under this condition, the production of the target protein was 4.5 times more than that in the medium without the additives. Overall, our approach to produce chimeric HFBII-VEGF-A165 and selectively capture it in ATPS is promising for large-scale protein production without laborious downstream processing.