Large-scale Production Of Heterologous Proteins Research Articles

A scalable suspension insect cell transfection method for production of baculoviruses with low amplification passages

Baculovirus expression vector systems (BEVS) have been widely used for production of recombinant proteins in insect cells. However, baculoviruses superinfection and repeated passages originate defective interfering particle (DIP) mutants, which is a limitation to a continuous large-scale production. Accordingly, a classical chemical transfection method performed on monolayer of Spodoptera frugiperda insect cells (Sf9) was modified to produce recombinant baculoviruses with high efficiency. Modifications consist to transfect exponentially growing cells in suspension after concentration by tenfold through centrifugation. Ten different constructions of recombinant baculoviruses with insert varying in size from 180 bp to 2,395 bp, were obtained through employment of the Bac-to-Bac expression system (ThermoFisher/Invitrogen). The transfection efficiency of the modified protocol varied from 45 to 57%, independent of the insert size, while classical method present transfection efficiency of 2 to 20%. After transfection of 6 × 106 cells, the recombinant baculoviruses titer obtained with modified method was about 2 × 107 pfu/ mL in a total volume of 12 mL, which is scalable to 24 liters of 1 × 108 pfu/ mL, after only two amplification rounds, contributing to improve large scale heterologous protein production in insect cells, with low amplification passages.

Optimization of Culture Medium for Large-Scale Production of Heterologous Proteins in Pichia pastoris to be used in Nanoscience and other Biotechnological Fields

Silver thiosulfate (STS) used in the post-harvest treatment of ethylene sensitive flowers is a potent producer of environmental damage because the silver cation (Ag+) remains in the soil and groundwater describes for long periods and can migrate to drinking water systems and cause health problems in humans. This study presents a novel treatment of wastewater contaminated with STS that includes three steps: i) oxidation of STS by bacteria isolated from sulfur springs of the Ecuadorian highlands which were added to artificial wastewater or wastewater containing STS from floriculture. The results indicate that approximately 60% of thiosulfate is oxidized to sulfate and 10% is absorbed by the bacteria at acidic pH, ii) biosorption of silver cation with fungi pellets of Cladosporium cladosporioides fungi. The adsorption tests show that the maximum adsorption capacity was 16 mg/g pellet using pellets prepared with fungi of 11 days of growth at pH 6, iii) regeneration of fungi pellets using 4N nitric acid solutions. With this acidic regenerant, it was recovered 69% of silver accumulated within the fungi pellets and the adsorption capacity of the regenerated fungi decreased to 15.4 mg Ag+/g in the second adsorption cycle.

Functional expression of plant membrane proteins in Lactococcus lactis.

The study of most membrane proteins remains challenging due to their hydrophobicity and their low natural abundance in cells. Lactococcus lactis, a Gram-positive lactic bacterium, has been traditionally used in food fermentations and is nowadays widely used in biotechnology for large-scale production of heterologous proteins. This system has been successfully used for the production of prokaryotic and eukaryotic membrane proteins. The purpose of this chapter is to provide detailed protocols for (1) the expression of plant peripheral or intrinsic membrane proteins and then for (2) their solubilization, from Lactococcus membranes, for further purification steps and biochemical characterization.

Multivariate modular engineering of the protein secretory pathway for production of heterologous glucose oxidase in Pichia pastoris

Limitations in protein production and secretion have been attributed to the inefficient folding rate of overexpressed proteins and the cellular response to the presence of overexpressed proteins in the endoplasmic reticulum (ER). In this study, we improved the yield of glucose oxidase (GOD) by manipulating genes involved in protein folding machinery and abnormal folding stress responses. First, genes with folding and secretion functions were used to modulate the folding rate of GOD in the ER and its secretion level in the cytoplasm. Next, the potential benefits of the ERAD elements were determined. Cellular resistance to ER derived stress was then strengthened by overexpressing the stress response gene GCN4. Furthermore, a module combination strategy, which co-expressed the SEC53, CNE1 and GCN4 genes, was employed to construct the Pichia pastoris strain S17. This increased the yield of GOD to 21.81g/L, with an activity of 1972.9U/mL, which were 2.53- and 5.11-fold higher, respectively, than the control strain. The work described here improved GOD production significantly, and the strategies employed in this study provide novel information for the large-scale production of heterologous proteins.

Cytoplasmic and extracellular expression of pharmaceutical-grade mycobacterial 65-kDa heat shock protein in Lactococcus lactis

Lactic acid bacteria (LAB) are an attractive and safe alternative for the expression of heterologous proteins, as they are nonpathogenic and endotoxin-free organisms. Lactococcus lactis, the LAB model organism, has been extensively employed in the biotechnology field for large-scale production of heterologous proteins, and its use as a "cell factory" has been widely studied. We have been particularly interested in the use of L. lactis for production of heat shock proteins (HSPs), which reportedly play important roles in the initiation of innate and adaptive immune responses. However, this activity has been questioned, as LPS contamination appears to be responsible for most, if not all, immunostimulatory activity of HSPs. In order to study the effect of pure HSPs on the immune system, we constructed recombinant L. lactis strains able to produce and properly address the Mycobacterium leprae 65-kDa HSP (Hsp65) to the cytoplasm or to the extracellular medium, using a xylose-induced expression system. Approximately 7 mg/L recombinant Hsp65 was secreted. Degradation products related to lactococcal HtrA activity were not observed, and the Limulus amebocyte lysate assay demonstrated that the amount of LPS in the recombinant Hsp65 preparations was 10-100 times lower than the permitted levels established by the U.S. Food and Drug Administration. These new L. lactis strains will allow investigation of the effects of M. leprae Hsp65 without the interference of LPS; consequently, they have potential for a variety of biotechnological, medical and therapeutic applications.

Lactococcus lactis, an Alternative System for Functional Expression of Peripheral and Intrinsic Arabidopsis Membrane Proteins

BackgroundDespite their functional and biotechnological importance, the study of membrane proteins remains difficult due to their hydrophobicity and their low natural abundance in cells. Furthermore, into established heterologous systems, these proteins are frequently only produced at very low levels, toxic and mis- or unfolded. Lactococcus lactis, a Gram-positive lactic bacterium, has been traditionally used in food fermentations. This expression system is also widely used in biotechnology for large-scale production of heterologous proteins. Various expression vectors, based either on constitutive or inducible promoters, are available for this system. While previously used to produce bacterial and eukaryotic membrane proteins, the ability of this system to produce plant membrane proteins was until now not tested.Methodology/Principal FindingsThe aim of this work was to test the expression, in Lactococcus lactis, of either peripheral or intrinsic Arabidopsis membrane proteins that could not be produced, or in too low amount, using more classical heterologous expression systems. In an effort to easily transfer genes from Gateway-based Arabidopsis cDNA libraries to the L. lactis expression vector pNZ8148, we first established a cloning strategy compatible with Gateway entry vectors. Interestingly, the six tested Arabidopsis membrane proteins could be produced, in Lactococcus lactis, at levels compatible with further biochemical analyses. We then successfully developed solubilization and purification processes for three of these proteins. Finally, we questioned the functionality of a peripheral and an intrinsic membrane protein, and demonstrated that both proteins were active when produced in this system.Conclusions/SignificanceAltogether, these data suggest that Lactococcus lactis might be an attractive system for the efficient and functional production of difficult plant membrane proteins.

Characteristics of Saccharomyces cerevisiae gal1Δ and gal1Δhxk2Δ mutants expressing recombinant proteins from the GAL promoter

Galactose can be used not only as an inducer of the GAL promoters, but also as a carbon source by Saccharomyces cerevisiae, which makes recombinant fermentation processes that use GAL promoters complicated and expensive. To overcome this problem during the cultivation of the recombinant strain expressing human serum albumin (HSA) from the GAL10 promoter, a gal1 Delta mutant strain was constructed and its induction kinetics investigated. As expected, the gal1 Delta strain did not use galactose, and showed high levels of HSA expression, even at extremely low galactose concentrations (0.05-0.1 g/L). However, the gal1 Delta strain produced much more ethanol, in a complex medium containing glucose, than the GAL1 strain. To improve the physiological properties of the gal1 Delta mutant strain as a host for heterologous protein production, a null mutation of either MIG1 or HXK2 was introduced into the gal1 Delta mutant strain, generating gal1 Delta mig1 Delta and gal1 Delta hxk2 Delta double strains. The gal1 Delta hxk2 Delta strain showed a decreased rate of ethanol synthesis, with an accelerated rate of ethanol consumption, compared to the gal1 Delta strain, whereas the gal1 Delta mig1 Delta strain showed similar patterns to the gal1 Delta strain. Furthermore, the gal1 Delta hxk2 Delta strain secreted much more recombinant proteins (HSA and HSA fusion proteins) than the other strains. The results suggest that the gal1 Delta hxk2 Delta strain would be useful for the large-scale production of heterologous proteins from the GAL10 promoter in S. cerevisiae.

Insect cells as a factory to produce adeno-associated virus type 2 vectors.

Recombinant adeno-associated viruses (rAAV) are produced transiently in mammalian cells usually by cotransfecting two or three plasmids containing AAV genes, adenovirus helper genes, and a vector genome. Expansion and transfection of adherent cells limit the scale of rAAV production. Efficient transfection is performed with cells on solid support media such as tissue culture plates. A large animal study or a human clinical trial may require 10(15) particles of vector, depending on dose. To generate this quantity of rAAV by transfection, more than 10(11) HEK293 cells may be needed, which would require about 5000 x 175 cm(2) flasks. The ability to scale up rAAV production by these methods severely restricts the commercialization and use of AAV vectors. A recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus is widely employed for large-scale production of heterologous proteins in cultured insect cells and may provide an attractive alternative. Toward this goal, we have explored the production of rAAV in invertebrate cells. Sf9 cells may be coinfected in suspension cultures with three recombinant baculoviruses (a Rep-baculovirus, a VP-baculovirus, and an AAV ITR vector genome baculovirus) and, 3 days later, rAAV is recovered. The particles produced are indistinguishable from 293 cell-produced rAAV, as determined on the basis of physical properties and biologic activities. Particles produced by either method were composed of similar proteins and nucleic acid. The yield of genome-containing particles produced per Sf9 cell approached 5 x 10(4), thus, 1000 ml of cultured Sf9 cells produced the equivalent of between 500 to 1000 x 175 cm(2) flasks of 293 cells. This robust system provides a simple, cost-effective method for AAV vector production.

Enhanced Expression of Cytochrome P450s from lac-Based Plasmids Using Lactose as the Inducer

The cytochrome P450 expression systems used in Escherichia coli are highly regulated and involve the use of the lac repressor to control expression. Induction in these systems utilizes the nonmetabolizable analog of lactose, isopropyl-β-d-thiogalactopyranoside (IPTG), which is the most expensive compound required for an E. coli expression system. To determine if the natural inducer lactose could be used to induce cytochrome P450 expression we examined the expression of three P450 enzymes in E. coli using two different expression systems, pTrc99A and the T7-based PET22b vector. For both systems lactose was found to induce expression of active P450 to concentrations that exceeded the levels achieved with IPTG. A 20-liter fermentation of a P450 expression system in the pTrc plasmid in which lactose was used as the inducer resulted in 2.4 μ mol P450/liter, with a total yield of 2 g of cytochrome P450. The use of lactose for protein expression in E. coli should be broadly useful for the inexpensive, large-scale production of heterologous proteins in E. coli.

Regulation of primary carbon metabolism in Kluyveromyces lactis.

In the recent past, through advances in development of genetic tools, the budding yeast Kluyveromyces lactis has become a model system for studies on molecular physiology of so-called "Nonconventional Yeasts." The regulation of primary carbon metabolism in K. lactis differs markedly from Saccharomyces cerevisiae and reflects the dominance of respiration over fermentation typical for the majority of yeasts. The absence of aerobic ethanol formation in this class of yeasts represents a major advantage for the "cell factory" concept and large-scale production of heterologous proteins in K. lactis cells is being applied successfully. First insight into the molecular basis for the different regulatory strategies is beginning to emerge from comparative studies on S. cerevisiae and K. lactis. The absence of glucose repression of respiration, a high capacity of respiratory enzymes and a tight regulation of glucose uptake in K. lactis are key factors determining physiological differences to S. cerevisiae. A striking discrepancy exists between the conservation of regulatory factors and the lack of evidence for their functional significance in K. lactis. On the other hand, structurally conserved factors were identified in K. lactis in a new regulatory context. It seems that different physiological responses result from modified interactions of similar molecular modules.

Synthesis of biotechnologically relevant heterologous proteins in filamentous fungi

Numerous eukaryotic expression systems have been developed for use in filamentous fungi since these microorganisms have a high capacity for protein production and secretion. Although commercial exploitation is still at an early stage, a greater understanding of post-translational protein modification and protein secretion mechanisms might help optimize large scale heterologous protein production systems. This is of particular relevance to those proteins that cannot be produced in sufficient quantities in other expression systems. Here, recently developed systems for synthesizing foreign polypeptides in filamentous fungi are reviewed, paying particular attention to the molecular aspects of regulating heterologous gene expression in different hosts. We also evaluate the application of such technology to pathway engineering and protein over-production.

Effects of cultivation conditions on the production of heterologous α-galactosidase by Kluyveromyces lactis

Growth conditions relevant for the large-scale production of heterologous proteins with yeasts were studied on a laboratory scale. A strain of Kluyveromyces lactis, containing 15 copies of an expression cassette encoding guar α-galactosidase integrated into its ribosomal DNA, was used as a model. By using urea as a nitrogen source, it was possible to produce active extracellular α-galactosidase in shake-flask cultures grown on a defined mineral medium. Inclusion of urea instead of ammonium sulphate prevented unwanted acidification of cultures. With urea-containing mineral medium, enzyme production in shake flasks was comparable to that in complex media containing peptone. In contrast, the presence of peptone was required to achieve high productivity in chemostat cultures. The low productivity in chemostat cultures growing on mineral media was not due to loss oft the expression cassette, since addition of peptone to such cultures resulted in an immediate high rate of α-galactosidase production. The discrepancy between the behaviour of shake-flask and chemostat cultures during growth on mineral medium illustrates the necessity of physiological studies for the scalling-up of heterologous protein production from laboratory to production scale.