Expression of a recombinant elastin‐like protein in pichia pastoris

The translation of highly repetitive gene sequences is often associated with reduced levels of protein expression and may be prone to mutational events. In this report, we describe a modified concatemerization strategy to construct a gene with enhanced sequence diversity that encodes a highly repetitive elastin‐like protein polymer for expression in Pichia pastoris. Specifically, degenerate oligonucleotides were used to create a monomer library, which after concatemerization yielded a genetically nonrepetitive DNA sequence that encoded identical pentapeptide repeat sequences. By limiting genetic repetition, the risk of genetic deletions, rearrangements, or premature termination errors during protein synthesis is minimized. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Considerations for the process development of insect‐derived antimicrobial peptide production

Antimicrobial peptides (AMPs) could evolve into new therapeutic lead molecules against multi‐resistant bacteria. As insects are a rich source of AMP, the identification and characterization of insect‐derived AMPs is particularly emphasized. One challenge of bringing these molecules into market, e.g., as a drug, is to develop a cost‐efficient large‐scale production process. Due to the fact that a direct AMP isolation from insects is not economical and that chemical synthesis is recommended for peptide sizes below 40 amino acids, a viable option is heterologous AMP production. Therefore, previous knowledge concerning the expression of larger proteins can be adapted, but due to the AMP nature (e.g., small size, bactericide) additional challenges have to be faced during up and downstream processing. Nonetheless the bottleneck for large‐scale AMP production is the same as for proteins; mainly the downstream process. This review introduces opportunities for insect‐derived AMP production, like the choice of the expression system (based on previously derived data), depending on the AMP nature, as well as new purification strategies like elastin‐like peptide/intein based purification strategies. All of these aspects are discussed with regard to large‐scale processes and costs. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:1–11, 2015     

Exploiting the intracellular compartmentalization characteristics of the S. cerevisiae host cell for enhancing primary purification of lipid‐envelope virus‐like particles

This article demonstrates how the intracellular compartmentalization of the S. cerevisiae host cell can be exploited to impart selectivity during the primary purification of lipid‐envelope virus‐like particles (VLPs). The hepatitis B surface antigen (HBsAg) was used as the VLP model in this study. Expressed HBsAg remain localized on the endoplasmic reticulum and the recovery process involves treating cell homogenate with a detergent for HBsAg liberation. In our proposed strategy, a centrifugation step is introduced immediately following cell disruption but prior to the addition of detergent to allow the elimination of bulk cytosolic contaminants in the supernatant, achieving ～70% reduction of contaminating yeast proteins, lipids, and nucleic acids. Recovery and subsequent treatment of the solids fraction with detergent then releases the HBsAg into a significantly enriched product stream with a yield of ～80%. The selectivity of this approach is further enhanced by operating under moderate homogenization pressure conditions (～400 bar). Observed improvements in the recovery of active HBsAg and reduction of contaminating host lipids were attributed to the low‐shear conditions experienced by the HBsAg product and reduced cell fragmentation, which led to lower coextraction of lipids during the detergent step. As a result of the cleaner process stream, the level of product capture during the loading stage of a downstream hydrophobic interaction chromatography stage increased by two‐fold leading to a concomitant increase in the chromatography step yield. The lower level of exposure to contaminants is also expected to improve column integrity and lifespan. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Regulation of dynamic polarity switching in bacteria by a Ras‐like G‐protein and its cognate GAP

The rod‐shaped cells of the bacterium Myxococcus xanthus move uni‐directionally and occasionally undergo reversals during which the leading/lagging polarity axis is inverted. Cellular reversals depend on pole‐to‐pole relocation of motility proteins that localize to the cell poles between reversals. We show that MglA is a Ras‐like G‐protein and acts as a nucleotide‐dependent molecular switch to regulate motility and that MglB represents a novel GTPase‐activating protein (GAP) family and is the cognate GAP of MglA. Between reversals, MglA/GTP is restricted to the leading and MglB to the lagging pole defining the leading/lagging polarity axis. For reversals, the Frz chemosensory system induces the relocation of MglA/GTP to the lagging pole causing an inversion of the leading/lagging polarity axis. MglA/GTP stimulates motility by establishing correct polarity of motility proteins between reversals and reversals by inducing their pole‐to‐pole relocation. Thus, the function of Ras‐like G‐proteins and their GAPs in regulating cell polarity is found not only in eukaryotes, but also conserved in bacteria.     

His‐tagged protein purification by metal‐chelate affinity extraction with nickel‐chelate reverse micelles

Di(2‐ethylhexyl) phosphoric acid (HDEHP) was used as a transition metal ion chelator and introduced to the nonionic reverse micellar system composed of equimolar Triton X‐45 and Span 80 at a total concentration of 30 mmol/L. Ni(II) ions were chelated to the HDEHP dimers in the reverse micelles, forming a complex denoted as Ni(II)R₂. The Ni(II)‐chelate reverse micelles were characterized for the purification of recombinant hexahistidine‐tagged enhanced green fluorescent protein (EGFP) expressed in Escherichia coli. The affinity binding of EGFP to Ni(II)R₂ was proved by investigation of the forward and back extraction behaviors of purified EGFP. Then, EGFP was purified with the affinity reverse micelles. It was found that the impurities in the feedstock impeded EGFP transfer to the reverse micelles, though they were little solubilized in the organic phase. The high specificity of the chelated Ni²⁺ ions toward the histidine tag led to the production of electrophoretically pure EGFP, which was similar to that purified by immobilized metal affinity chromatography. A two‐stage purification by the metal‐chelate affinity extraction gave rise to 87% recovery of EGFP. Fluorescence spectrum analysis suggests the preservation of native protein structure after the separation process, indicating the system was promising for protein purification. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Structural insights into oligomerization and mitochondrial remodelling of dynamin 1‐like protein

Dynamin 1‐like protein (DNM1L) mediates fission of mitochondria and peroxisomes, and dysfunction of DNM1L has been implicated in several neurological disorders. To study the molecular basis of mitochondrial remodelling, we determined the crystal structure of DNM1L that is comprised of a G domain, a bundle signalling element and a stalk. DNM1L assembled via a central stalk interface, and mutations in this interface disrupted dimerization and interfered with membrane binding and mitochondrial targeting. Two sequence stretches at the tip of the stalk were shown to be required for ordered assembly of DNM1L on membranes and its function in mitochondrial fission. In the crystals, DNM1L dimers further assembled via a second, previously undescribed, stalk interface to form a linear filament. Mutations in this interface interfered with liposome tubulation and mitochondrial remodelling. Based on these results and electron microscopy reconstructions, we propose an oligomerization mode for DNM1L which differs from that of dynamin and might be adapted to the remodelling of mitochondria.     

Cell‐free protein synthesis and purification of human dopamine D2 receptor long isoform

The human dopamine D2 receptor long isoform (D2L) has significant implications in neurological and neuropsychiatric disorders such as Parkinson's disease and schizophrenia. Detailed structural knowledge of this receptor is limited owing to its highly hydrophobic nature, which leads to protein aggregation and host toxicity when expressed in cellular systems. The newly emerging field of cell‐free protein expression presents numerous advantages to overcome these challenges. This system utilizes protein synthesis machinery and exogenous DNA to synthesize functional proteins outside of intact cells. This study utilizes two different cell‐free systems for the synthesis of human dopamine D2L receptor. These include the Escherichia coli lysate‐based system and the wheat‐germ lysate‐based system. The bacterial cell‐free method used pET 100/D‐TOPO vector to synthesize hexa‐histidine‐tagged D2L receptor using a dialysis bag system; the resulting protein was purified using nickel‐nitrilotriacetic acid affinity resin. The wheat germ system used pEU–glutathione‐S‐transferase (GST) vector to synthesize GST‐tagged D2L receptor using a bilayer translation method; the resulting protein was purified using a GST affinity resin. The presence and binding capacity of the synthesized D2L receptor was confirmed by immunoblotting and radioligand competition assays, respectively. Additionally, in‐gel protein sequencing via Nano LC‐MS/MS was used to confirm protein synthesis via the wheat germ system. The results showed both systems to synthesize microgram quantities of the receptor. Improved expression of this highly challenging protein can improve research and understanding of the human dopamine D2L receptor. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:601–608, 2013     

Ontogeny of angiopoietin‐like protein 1, 2, 3, 4, 5, and 7 genes during chick embryonic development

Angiopoietin‐like proteins (ANGPTLs) are secreted proteins possessing an amino‐terminal coiled‐coil domain and a carboxyl‐terminal fibrinogen‐like domain and are known as angiogenic factors. Several members of ANGPTLs also regulate lipid metabolism independently of angiogenic effects, but most of their functions during vertebrate development are not demonstrated. To ascertain their developmental functions, we examined the expression patterns of Angptl1, 2, 3, 4, 5, and 7 orthologues during chick development using whole‐mount in situ hybridization. Angptl1 was first detected at embryonic day 3 (E3) in the somite. At E4, Angptl1 was expressed in somite‐derivatives and limb mesenchyme. Angptl2 was first detected at E3 in the hindbrain. At E4, Angptl2 was expressed in neuroepithelium of forebrain and hindbrain and partly in the heart. Angptl3 was first detected at E3 and continued to be expressed in the liver and yolk sac at E4. Angptl4 was first detected at E3 in the somites and liver. At E4, Angptl4 was also observed in the heart. Angptl5 was not detected in these developmental stages. Angptl7 was first detected at E3 in the ectoderm overlying the lenses of the eyes. At E4, Angptl7 was specifically expressed in cornea. These data suggest that each member of the ANGPTL family could be related to angiogenesis during various organogeneses of the developing chick embryo.     

Prion‐like Doppel gene polymorphisms and scrapie susceptibility in portuguese sheep breeds

The establishment of an association between prion protein gene (PRNP) polymorphisms and scrapie susceptibility in sheep has enabled the development of breeding programmes to increase scrapie resistance in the European Union. Intense selection for PRNP genotype may lead to correlated selection for genes linked to PRNP. We intended to investigate if any association exists between genetic variation in prion‐like protein Doppel gene (PRND) and scrapie susceptibility, determined through PRNP genotyping. Sampling included 460 sheep from eight Portuguese breeds and the PRND gene coding region was analysed by multiple restriction fragment‐single strand conformation polymorphism (MRF‐SSCP), whereas PRNP genotyping was carried out by primer extension. A synonymous substitution (c.78G>A) was detected in codon 26 of the PRND gene, in all breeds except Churra Mondegueira. Linkage disequilibrium was found between the PRND and PRNP loci (P = 0.000). Specifically, PRND was monomorphic in the 45 animals with the more resistant ARR/ARR PRNP genotype (P = 0.003), whereas a higher frequency of PRND heterozygotes (GA) was associated with ARQ/AHQ (P = 0.029). These results constitute preliminary evidence of an association between a polymorphism in the PRND gene and scrapie susceptibility, and indicate that the possibility of undesirable consequences from widespread selection for PRNP genotype on genetic diversity and reproduction traits needs to be further investigated.     

Shedding of glycan‐modifying enzymes by signal peptide peptidase‐like 3 (SPPL3) regulates cellular N‐glycosylation

Protein N‐glycosylation is involved in a variety of physiological and pathophysiological processes such as autoimmunity, tumour progression and metastasis. Signal peptide peptidase‐like 3 (SPPL3) is an intramembrane‐cleaving aspartyl protease of the GxGD type. Its physiological function, however, has remained enigmatic, since presently no physiological substrates have been identified. We demonstrate that SPPL3 alters the pattern of cellular N‐glycosylation by triggering the proteolytic release of active site‐containing ectodomains of glycosidases and glycosyltransferases such as N‐acetylglucosaminyltransferase V, β‐1,3 N‐acetylglucosaminyltransferase 1 and β‐1,4 galactosyltransferase 1. Cleavage of these enzymes leads to a reduction in their cellular activity. In line with that, reduced expression of SPPL3 results in a hyperglycosylation phenotype, whereas elevated SPPL3 expression causes hypoglycosylation. Thus, SPPL3 plays a central role in an evolutionary highly conserved post‐translational process in eukaryotes.     

Structural basis for receptor recognition and pore formation of a zebrafish aerolysin‐like protein

Various aerolysin‐like pore‐forming proteins have been identified from bacteria to vertebrates. However, the mechanism of receptor recognition and/or pore formation of the eukaryotic members remains unknown. Here, we present the first crystal and electron microscopy structures of a vertebrate aerolysin‐like protein from Danio rerio, termed Dln1, before and after pore formation. Each subunit of Dln1 dimer comprises a β‐prism lectin module followed by an aerolysin module. Specific binding of the lectin module toward high‐mannose glycans triggers drastic conformational changes of the aerolysin module in a pH‐dependent manner, ultimately resulting in the formation of a membrane‐bound octameric pore. Structural analyses combined with computational simulations and biochemical assays suggest a pore‐forming process with an activation mechanism distinct from the previously characterized bacterial members. Moreover, Dln1 and its homologs are ubiquitously distributed in bony fishes and lamprey, suggesting a novel fish‐specific defense molecule.     

High‐throughput mAb expression and purification platform based on transient CHO

A high‐cell‐density transient transfection system was recently developed in our laboratory based on a CHO‐GS‐KO cell line. This method yields monoclonal antibody titers up to 350 mg/L from a simple 7‐day process, in volumes ranging from 2 mL to 2 L. By performing transfections in 24‐deep‐well plates, a large number of mAbs can be expressed simultaneously. We coupled this new high‐throughput transfection process to a semiautomated protein A purification process. Using a Biomek FX^p liquid handling robot, up to 72 unique mAbs can be simultaneously purified. Our primary goal was to obtain >0.25 mg of purified mAb at a concentration of >0.5 mg/mL, without any concentration or buffer‐exchange steps. We optimized both the batch‐binding and the batch elution steps. The length of the batch‐binding step was important to minimize mAb losses in the flowthrough fraction. The elution step proved to be challenging to simultaneously maximize protein recovery and protein concentration. We designed a variable volume elution strategy based on the average supernatant titer. Finally, we present two case studies. In the first study, we produced 56 affinity maturation mAb variants at an average yield of 0.33 ± 0.05 mg (average concentration of 0.65 ± 0.10 mg/mL). In a second study, we produced 42 unique mAbs, from an early‐stage discovery effort, at an average yield of 0.79 ± 0.31 mg (average concentration of 1.59 ± 0.63 mg/mL). The combination of parallel high‐yielding transient transfection and semiautomated high‐throughput protein A purification represents a valuable mAb drug discovery tool. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:239–247, 2015     

Elastin‐like‐polypeptide based fusion proteins for osteogenic factor delivery in bone healing

Modern treatments of bone injuries and diseases are becoming increasingly dependent on the usage of growth factors to stimulate bone growth. Bone morphogenetic protein‐2 (BMP‐2), a potent osteogenic inductive protein, exhibits promising results in treatment models, but recently has had its practical efficacy questioned due to the lack of local retention, ectopic bone formation, and potentially lethal inflammation. Where a new delivery technique of the BMP‐2 is necessary, here we demonstrate the viability of an elastin‐like peptide (ELP) fusion protein containing BMP‐2 for delivery of the BMP‐2. This fusion protein retains the performance characteristics of both the BMP‐2 and ELP. The fusion protein was found to induce osteogenic differentiation of mesenchymal stem cells as evidenced by the production of alkaline phosphatase and extracellular calcium deposits in response to treatment by the fusion protein. Retention of the ELPs inverse phase transition property has allowed for expression of the fusion protein within a bacterial host (such as Escherichia coli) and easy and rapid purification using inverse transition cycling. The fusion protein formed self‐aggregating nanoparticles at human‐body temperature. The data collected suggests the viability of these fusion protein nanoparticles as a dosage‐efficient and location‐precise noncytotoxic delivery vehicle for BMP‐2 in bone treatment. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1029–1037, 2016     

Constraints imposed by non‐functional protein–protein interactions on gene expression and proteome size

Crowded intracellular environments present a challenge for proteins to form functional specific complexes while reducing non‐functional interactions with promiscuous non‐functional partners. Here we show how the need to minimize the waste of resources to non‐functional interactions limits the proteome diversity and the average concentration of co‐expressed and co‐localized proteins. Using the results of high‐throughput Yeast 2‐Hybrid experiments, we estimate the characteristic strength of non‐functional protein–protein interactions. By combining these data with the strengths of specific interactions, we assess the fraction of time proteins spend tied up in non‐functional interactions as a function of their overall concentration. This allows us to sketch the phase diagram for baker's yeast cells using the experimentally measured concentrations and subcellular localization of their proteins. The positions of yeast compartments on the phase diagram are consistent with our hypothesis that the yeast proteome has evolved to operate closely to the upper limit of its size, whereas keeping individual protein concentrations sufficiently low to reduce non‐functional interactions. These findings have implication for conceptual understanding of intracellular compartmentalization, multicellularity and differentiation.     

Optimization of norovirus virus‐like particle production in Pichia pastoris using a real‐time near‐infrared bioprocess monitor

The production of norovirus virus‐like particles (NoV VLPs) displaying NY‐ESO‐1 cancer testis antigen in Pichia pastoris BG11 Mut⁺ has been enhanced through feed‐strategy optimization using a near‐infrared bioprocess monitor (RTBio^® Bioprocess Monitor, ASL Analytical, Inc.), capable of monitoring and controlling the concentrations of glycerol and methanol in real‐time. The production of NoV VLPs displaying NY‐ESO‐1 in P. pastoris has potential as a novel cancer vaccine platform. Optimization of the growth conditions resulted in an almost two‐fold increase in the expression levels in the fermentation supernatant of P. pastoris as compared to the starting conditions. We investigated the effect of methanol concentration, batch phase time, and batch to induction transition on NoV VLP‐NY‐ESO‐1 production. The optimized process included a glycerol transition phase during the first 2 h of induction and a methanol concentration set point of 4 g L^?1 during induction. Utilizing the bioprocess monitor to control the glycerol and methanol concentrations during induction resulted in a maximum NoV VP1‐NY‐ESO‐1 yield of 0.85 g L^?1. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:518–526, 2016     

A mutational analysis of the cytosolic domain of the tomato Cf‐9 disease‐resistance protein shows that membrane‐proximal residues are important for Avr9‐dependent necrosis

The tomato Cf‐9 gene encodes a membrane‐anchored glycoprotein that imparts race‐specific resistance against the tomato leaf mould fungus Cladosporium fulvum in response to the avirulence protein Avr9. Although the N‐terminal half of the extracellular leucine‐rich repeat (eLRR) domain of the Cf‐9 protein determines its specificity for Avr9, the C‐terminal half, including its small cytosolic domain, is postulated to be involved in signalling. The cytosolic domain of Cf‐9 carries several residues that are potential sites for ubiquitinylation or phosphorylation, or signals for endocytic uptake. A targeted mutagenesis approach was employed to investigate the roles of these residues and cellular processes in Avr9‐dependent necrosis triggered by Cf‐9. Our results indicate that the membrane‐proximal region of the cytosolic domain of Cf‐9 plays an important role in Cf‐9‐mediated necrosis, and two amino acids within this region, a threonine (T835) and a proline (P838), are particularly important for Cf‐9 function. An alanine mutation of T835 had no effect on Cf‐9 function, but an aspartic acid mutation, which mimics phosphorylation, reduced Cf‐9 function. We therefore postulate that phosphorylation/de‐phosphorylation of T835 could act as a molecular switch to determine whether Cf‐9 is in a primed or inactive state. Yeast two‐hybrid analysis was used to show that the cytosolic domain of Cf‐9 interacts with the cytosolic domain of tomato VAP27. This interaction could be disrupted by an alanine mutation of P838, whereas interaction with CITRX remained unaffected. We therefore postulate that a proline‐induced kink in the membrane‐proximal region of the cytosolic domain of Cf‐9 may be important for interaction with VAP27, which may, in turn, be important for Cf‐9 function.     

A practical strategy for using miniature chromatography columns in a standardized high‐throughput workflow for purification development of monoclonal antibodies

The emergence of monoclonal antibody (mAb) therapies has created a need for faster and more efficient bioprocess development strategies in order to meet timeline and material demands. In this work, a high‐throughput process development (HTPD) strategy implementing several high‐throughput chromatography purification techniques is described. Namely, batch incubations are used to scout feasible operating conditions, miniature columns are then used to determine separation of impurities, and, finally, a limited number of lab scale columns are tested to confirm the conditions identified using high‐throughput techniques and to provide a path toward large scale processing. This multistep approach builds upon previous HTPD work by combining, in a unique sequential fashion, the flexibility and throughput of batch incubations with the increased separation characteristics for the packed bed format of miniature columns. Additionally, in order to assess the applicability of using miniature columns in this workflow, transport considerations were compared with traditional lab scale columns, and performances were mapped for the two techniques. The high‐throughput strategy was utilized to determine optimal operating conditions with two different types of resins for a difficult separation of a mAb monomer from aggregates. Other more detailed prediction models are cited, but the intent of this work was to use high‐throughput strategies as a general guide for scaling and assessing operating space rather than as a precise model to exactly predict performance. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:626–635, 2014