Levels Of Recombinant Protein Production Research Articles

Efficient CRISPR/Cas9-Mediated BAX Gene Ablation in CHO Cells To Impair Apoptosis and Enhance Recombinant Protein Production.

Despite recent advances in recombinant biotherapeutics production using CHO cells, their productivity remains lower than industrial needs, mainly due to apoptosis. Present study aimed to exploit CRISPR/Cas9 technology to specifically disrupt the BAX gene to attenuate apoptosis in recombinant Chinese hamster's ovary cells producing erythropoietin. The STRING database was used to identify the key pro-apoptotic genes to be modified by CRISPR/Cas9 technique. The single guide RNAs (sgRNAs) targeting identified gene (BAX) were designed, and CHO cells were then transfected with vectors. Afterward, changes in the expression of the Bax gene and consequent production rates of erythropoietin were investigated in manipulated cells, even in the presence of an apoptosis inducer agent, oleuropein. BAX disruption significantly prolonged cell viability and increased proliferation rate in manipulated clones (152%, P-value = 0.0002). This strategy reduced the levels of Bax protein expression in manipulated cells by more than 4.3-fold (P-value <0.0001). The Bax-8 manipulated cells displayed higher threshold tolerance to the stress and consequence apoptosis compared to the control group. Also, they exhibited a higher IC50 compared to the control in the presence of oleuropein (5095 µM.ml-1 Vs. 2505 µM.ml-1). We found a significant increase in recombinant protein production levels in manipulated cells, even in the presence of 1,000 µM oleuropein compared to the control cell line (p-value=0.0002). CRISPR/Cas9 assisted BAX gene ablation is promising to improve erythropoietin production in CHO cells via engineering anti-apoptotic genes. Therefore, exploiting genome editing tools such as CRISPR/Cas9 has been proposed to develop host cells that result in a safe, feasible, and robust manufacturing operation with a yield that meets the industrial requirements.

Hydrodynamic Effects on Biofilm Development and Recombinant Protein Expression.

Hydrodynamics play an important role in the rate of cell attachment and nutrient and oxygen transfer, which can affect biofilm development and the level of recombinant protein production. In the present study, the effects of different flow conditions on the development of Escherichia coli biofilms and the expression of a model recombinant protein (enhanced green fluorescent protein, eGFP) were examined. Planktonic and biofilm cells were grown at two different flow rates in a recirculating flow cell system for 7 days: 255 and 128 L h−1 (corresponding to a Reynolds number of 4600 and 2300, respectively). The fluorometric analysis showed that the specific eGFP production was higher in biofilms than in planktonic cells under both hydrodynamic conditions (3-fold higher for 255 L h−1 and 2-fold higher for 128 L h−1). In the biofilm cells, the percentage of eGFP-expressing cells was on average 52% higher at a flow rate of 255 L h−1. Furthermore, a higher plasmid copy number (PCN) was obtained for the highest flow rate for both planktonic (244 PCN/cell versus 118 PCN/cell) and biofilm cells (43 PCN/cell versus 29 PCN/cell). The results suggested that higher flow velocities promoted eGFP expression in E. coli biofilms.

Innovative Bioprocess Strategies Combining Physiological Control and Strain Engineering of Pichia pastoris to Improve Recombinant Protein Production.

The combination of strain and bioprocess engineering strategies should be considered to obtain the highest levels of recombinant protein production (RPP) while assuring product quality and process reproducibility of heterologous products. In this work, two complementary approaches were investigated to improve bioprocess efficiency based on the yeast P. pastoris. Firstly, the performance of two Candida rugosa lipase 1 producer clones with different gene dosage under the regulation of the constitutive P GAP were compared in chemostat cultures with different oxygen-limiting conditions. Secondly, hypoxic conditions in carbon-limited fed-batch cultures were applied by means of a physiological control based on the respiratory quotient (RQ). Stirring rate was selected to maintain RQ between 1.4 and 1.6, since it was found to be the most favorable in chemostat. As the major outcome, between 2-fold and 4-fold higher specific production rate (q P ) values were observed when comparing multicopy clone (MCC) and single-copy clone (SCC), both in chemostat and fed-batch. Additionally, when applying oxygen limitation, between 1.5-fold and 3-fold higher q P values were obtained compared with normoxic conditions. Thus, notable increases of up to 9-fold in the production rates were reached. Furthermore, transcriptional analysis of certain key genes related to RPP and central carbon metabolism were performed. Results seem to indicate the presence of a limitation in post-transcriptional protein processing steps and a possible transcription attenuation of the target gene in the strains with high gene dosage. The entire approach, including both strain and bioprocess engineering, represents a relevant novelty involving physiological control in Pichia cell factory and is of crucial interest in bioprocess optimization, boosting RPP, allowing bioproducts to be economically competitive in the market, and helping develop the bioeconomy.

Synthesis and biological effects of small molecule enhancers for improved recombinant protein production in plant cell cultures.

Histone deacetylases are involved in chromatin remodelling and thus play a vital role in the epigenetic regulation of gene expression. HDAC inhibitors alter the acetylation status of histone and non-histone proteins to regulate various cellular events such as transcription. Novel HDAC inhibitors were designed and synthesised to promote higher levels of recombinant protein production in tobacco cell cultures. The effect of these chemical enhancers on the epigenetic profiles in plant cells has been evaluated by molecular docking, in vitro and in vivo studies. The addition of these novel enhancers led to an increase in histone H3 acetylation levels that promoted an increase in the accumulation levels of the recombinant protein in cell culture. These results can pave the way for the application of these enhancers to improve the production of high value products in plant cell based systems.

Expression of recombinant enhanced green fluorescent protein provides insight into foreign gene-expression differences between Mut+ and MutS strains of Pichia pastoris.

Pichia pastoris is a very popular yeast for recombinant protein production, mainly due to the strong, methanol-inducible PAOX1 promoter. Methanol induction however poses several drawbacks. One approach to improve processes is to use MutS strains with reduced methanol catabolic ability. Various reports claim that MutS allows higher recombinant protein production levels than Mut+ but scarcely elaborate on reasons for differences. In this study, enhanced green fluorescent protein was used as a PAOX1 -driven reporter for the investigation of expression differences between Mut+ and MutS strains. Mut+ exhibited higher responses to methanol, with faster growth (0.07 vs. 0.01hr-1 ) and higher consumption of methanol (2.25 vs. 1.81mmol/gDCW .hr) and oxygen (2.2 vs. 0.66mmol/gDCW .hr) than MutS. Mut+ yielded more biomass than MutS (2.3 vs. 1.3gDCW /L), and carbon dioxide analysis of bioreactor off-gas suggested that considerable amounts of methanol were consumed by Mut+ via the dissimilatory pathway. In contrast, it was demonstrated that the MutS population switched to an induced state more rapidly than Mut+. In addition, MutS exhibited 3.4-fold higher fluorescence levels per cell (77,509 vs. 23,783SFU) indicative of higher recombinant protein production. The findings were verified by similar results obtained during the expression of a lipase. Based on the differences in response to methanol versus recombinant protein production, it was proposed that higher energy availability occurs in MutS for recombinant protein synthesis, contrary to Mut+ that uses the energy to maintain high levels of methanol catabolic pathways and biomass production.

Proteomic analysis of CHO cell lines producing high and low quantities of a recombinant antibody before and after selection with methotrexate

High levels of recombinant protein production in Chinese hamster ovary (CHO) cells can be achieved by amplification of transgenes using the dihydrofolate reductase/methotrexate (DHFR/MTX) system. With the aim to identify predictive markers enabling the preselection of suitable high producing clones we investigated the impact of MTX-based gene amplification on two CHO cells lines producing different levels of a human monoclonal antibody by carrying out a comparative proteome analysis. The difference in antibody yield between the high and low producer was 15-fold before and 245-fold after MTX selection. Difference in-gel electrophoresis of samples from before and after MTX selection revealed 17 unique proteins that were differentially expressed between the high and low productivity lines. Of these, five proteins were differently expressed before MTX selection, representing potential markers for productivity prior to selection and for engineering processes to generate novel CHO cell line with the desirable high productivity phenotype. Fifteen proteins were differently expressed between high and low producer after MTX selection. We further found that MTX selection induced more changes in the proteome of the low producer compared to the high producer.

Improving the baculovirus expression vector system with vankyrin‐enhanced technology

The baculovirus expression vector system (BEVS) is a widely used platform for the production of recombinant eukaryotic proteins. However, the BEVS has limitations in comparison to other higher eukaryotic expression systems. First, the insect cell lines used in the BEVS cannot produce glycoproteins with complex‐type N‐glycosylation patterns. Second, protein production is limited as cells die and lyse in response to baculovirus infection. To delay cell death and lysis, we transformed several insect cell lines with an expression plasmid harboring a vankyrin gene (P‐vank‐1), which encodes an anti‐apoptotic protein. Specifically, we transformed Sf9 cells, Trichoplusia ni High FiveTM cells, and SfSWT‐4 cells, which can produce glycoproteins with complex‐type N‐glycosylation patterns. The latter was included with the aim to increase production of glycoproteins with complex N‐glycans, thereby overcoming the two aforementioned limitations of the BEVS. To further increase vankyrin expression levels and further delay cell death, we also modified baculovirus vectors with the P‐vank‐1 gene. We found that cell lysis was delayed and recombinant glycoprotein yield increased when SfSWT‐4 cells were infected with a vankyrin‐encoding baculovirus. A synergistic effect in elevated levels of recombinant protein production was observed when vankyrin‐expressing cells were combined with a vankyrin‐encoding baculovirus. These effects were observed with various model proteins including medically relevant therapeutic proteins. In summary, we found that cell lysis could be delayed and recombinant protein yields could be increased by using cell lines constitutively expressing vankyrin or vankyrin‐encoding baculovirus vectors. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 33:1496–1507, 2017

A novel expression system for lytic polysaccharide monooxygenases

Lytic polysaccharide monooxygenases (LPMOs) are key enzymatic players of lignocellulosic biomass degradation processes. As such, they have been introduced in cellulolytic cocktails for more efficient and less expensive lignocellulose saccharification. The recombinant production of LPMOs in bacteria for scientific investigations using vectors typically based on the T7 and lacUV5 promoters has been hampered by low yields. Reasons for this have been catabolite repression when producing the proteins in defined media with glucose as the sole carbon source, as well as the lack of an inducible expression system that allows controlled production of LPMOs that are correctly processed during translocation to the periplasmic space. A cassette vector design containing the XylS/Pm system was constructed and evaluated, showing that the expression cassette could easily be used for exchanging LPMO coding genes with or without signal sequences. The cassette was shown to reliably produce mature (translocated) LPMOs under controlled conditions that were achieved by using a low dosage (0.1 mM) of the Pm inducer m-toluic acid and a low (16 °C) cultivation temperature after induction. Furthermore, the signal sequences of five bacterial LPMOs were tested, and the signal sequence of LPMO10A from Serratia marcescens was found to give highest levels of recombinant protein production and translocation. The LPMO expression cassette was also evaluated in cultivations using defined media with glucose as the sole carbon source with a product yield of 7–22 mg per L of culture in shaking flasks. The integrity of the recombinant proteins were analyzed using NMR spectroscopy, showing that the system produced correctly processed and folded LPMOs. Finally, high cell-density cultivations of the recombinant strains were carried out, demonstrating stable protein production levels at similar relative yields (42–1298 mg per L of culture; 3.8–11.6 mg per OD600nm unit) as in shaking flasks, and showing the scale-up potential of the system.

The Use of Transcription Terminators to Generate Transgenic Lines of Chinese Hamster Ovary Cells (CHO) with Stable and High Level of Reporter Gene Expression

Mammalian cell lines are widely used to produce recombinant proteins. Stable transgenic cell lines usually contain many insertions of the expression vector in one genomic region. Transcription through transgene can be one of the reasons for target gene repression after prolonged cultivation of cell lines. In the present work, we used the known transcription terminators from the SV40 virus, as well as the human β- and γ-globin genes, to prevent transcription through transgene. The transcription terminators were shown to increase and stabilize the expression of the EGFP reporter gene in transgenic lines of Chinese hamster ovary (CHO) cells. Hence, transcription terminators can be used to create stable mammalian cells with a high and stable level of recombinant protein production.

Cloning and Expression of Fusion (F) and Haemagglutinin-neuraminidase (HN) Epitopes in Hairy Roots of Tobacco (Nicotiana tabaccum) as a Step Toward Developing a Candidate Recombinant Vaccine Against Newcastle Disease

Newcastle is a significant avian disease continuing to cause considerable loss. Developments in genetic engineering have led to plant-based platforms for human and animal vaccine production. Recombinant vaccine production in hairy root systems have several advantages over stable expression in whole plants, including high growth rates, ready genetic manipulations, high levels of recombinant protein production, and the potential for bioreactor culture. In an attempt to develop a recombinant vaccine in hairy roots, the sequences encoding fusion (F) and haemagglutinin-neuraminidase (HN) epitopes of Newcastle disease virus were cloned in pBI121 expression vector which was then transferred into leaf disks of tobacco (Nicotiana tabaccum) 'Turkish' cultivar by means of Agrobacterium rhizogenes. Hairy roots developed on MS medium containing 50 mg/L kanamycin and 30 mg/L meropenem. Incorporation of the heterologous gene in the genome of hairy roots was confirmed by PCR. Expression analyses were performed by real-time PCR at transcription level and by dot-blot and ELISA assays at translation level, all confirming the expression of the heterologous gene and production of the recombinant protein.

Transcriptional and post-transcriptional limitations of high-yielding, PEI-mediated transient transfection with CHO and HEK-293E cells.

Transient gene expression (TGE) in human embryonic kidney (HEK-293) and Chinese hamster ovary (CHO) cells is a well-established technology for the rapid generation of recombinant proteins. Although the maximum TGE yields have reached 1 g/L or more, the amount of plasmid DNA (pDNA) required for transfection remains high. Although greater than 10(3) copies of pDNA are present per transfected cell, protein yields are still lower than those achieved in recombinant cell lines with only one or a few copies of the transgene. This indicates a clear limitation to TGE in terms of the maximum level of recombinant protein production. In this study, we investigated the limitations to high-yielding TGE processes with CHO and HEK-293E cells using a monoclonal antibody as a model protein. For either cell host, both the intracellular and intranuclear pDNA levels increased linearly with the amount of pDNA added to the culture. In contrast, transgene mRNA accumulation reached a plateau as the intranuclear pDNA amount increased, suggesting a limitation in pDNA transcription. A post-transcriptional limitation to TGE yields was revealed by calculating the amount of antibody produced per transgene mRNA (mRNA utilization). For both hosts the transgene mRNA utilization decreased dramatically when transfected pDNA amounts increased beyond the level giving the maximum protein yield. The post-transcriptional limitation did not appear to be due to bottlenecks in antibody assembly or secretion, suggesting that transgene mRNA translation may be limiting. The results show that TGE yields are not limited by pDNA delivery into the nuclei, but in pDNA and transgene mRNA utilization.

New Promoters to Improve Heterologous Protein Production in Aspergillus vadensis

Aspergillus is a widely used host organism for the industrial production of homologous and heterologous proteins. Although Aspergillus niger is most commonly used, a close relative of this species, Aspergillus vadensis, has been suggested as a suitable and more favourable alternative due in part, to the low levels of extracellular proteases which it produces. Despite much progress being made in the hyper production of homologous proteins, the yields obtained for heterologous proteins have still not reached a comparable level. Genetic strategies, including the development of strong constitutional promoters have been shown to lead to an increase in the levels of recombinant protein production. In this study, six novel constitutional promoters from A. niger (pef1α, ptktA, pef1β, ptal1, pcetA and ppgkA) and a further five from A. vadensis (pef1α, prps31, pgpdA, pubi1 and poliC) were tested in A. vadensis using a gene encoding a secreted arabinofuranosidase from Fusarium oxysporum as a reporter for heterologous protein production. Remarkably, 9 of the 11 promoter constructs tested resulted in higher ABF activity than the commonly used gpdA promoter. While this could partly be assigned to the number of copies of the expression cassette in the transformants, clear differences in productivity of the promoters could be observed.

Rapid and massive green fluorescent protein production leads to formation of protein Y-bodies in plant cells

Although high level of recombinant protein production can be achieved via transient expression in plant cells, the mechanism by which tolerance to the presence of recombinant protein is acquired remains unclear. Here we show that green fluorescent protein (GFP) encoded by an intron-optimized tobacco mosaic viral vector formed large membraneless GFP bodies called Y-bodies that demonstrated mainly perinuclear localization. The Y-bodies were heterogeneous in size, approaching the size of the cell nucleus. Experiments with extracted GFP and live cell imaging showed that Y-bodies included actively fluorescent, non-aggregated, tightly packed GFP molecules. The plant cells probably formed Y-bodies to exclude the recombinant protein from normal physiological turnover.

Susceptibility to AcMNPV and expression of recombinant proteins by a novel cell clone derived from a Trichoplusia ni QAU-BTI-Tn9-4s cell line

It is well known that Tn5B1-4 (commercially known as the High Five) cell line is highly susceptible to baculovirus and provides superior production of recombinant proteins when compared to other insect cell lines. But the characteristics of the cell line do not always remain stable and may change upon continuous passage. Recently an alphanodavirus, named Tn5 Cell Line Virus (or TNCL Virus), was identified in High Five cells in particular. Therefore, we established a new cell line, QB-Tn9-4s, from Trichoplusia ni, which was determined to be free of TNCL virus by RT-PCR analysis. In this paper, we describe the development of a novel cell clone, QB-CL-B, from a low passage QB-Tn9-4s cell line and report its susceptibility to AcMNPV, and the level of recombinant protein production. This cell clone was similar to its parental cells QB-Tn9-4s and Tn5B1-4 cells in morphology and growth rate; although it also showed approximately the same responses to AcMNPV infection and production of occlusion bodies, there were higher levels of recombinant protein production in comparison to QB-Tn9-4s (parental cells) and High5 cells.

Fluorinert, an oxygen carrier, improves cell culture performance in deep square 96‐well plates by facilitating oxygen transfer

In bioprocess development, the 96-well plate format has been widely used for high-throughput screening of production cell line or culture conditions. However, suspension cell cultures in conventional 96-well plates often fail to reach high cell density under normal agitation presumably due to constraints in oxygen transfer. Although more vigorous agitation can improve gas transfer in 96-well plate format, it often requires specialized instruments. In this report, we employed Fluorinert, a biologically inert perfluorocarbon, to improve oxygen transfer in 96-well plate and to enable the growth of a Chinese Hamster Ovary cell line expressing a recombinant monoclonal antibody. When different amounts of Fluorinert were added to the cell culture medium, a dose-dependent improvement in cell growth was observed in both conventional and deep square 96-well plates. When sufficient Fluorinert was present in the culture, the cell growth rate, the peak cell density, and recombinant protein production levels achieved in deep square 96-wells were comparable to cultures in ventilated shake flasks. Although Fluorinert is known to dissolve gases such as oxygen and CO(2), it does not dissolve nor extract medium components, such as glucose, lactate, or amino acids. We conclude that mixing Fluorinert with culture media is a suitable model for miniaturization of cell line development and process optimization. Proper cell growth and cellular productivity can be obtained with a standard shaker without the need for any additional aeration or vigorous agitation.

Identification of the limitations on recombinant gene expression in CHO cell lines with varying luciferase production rates

Mammalian cell lines are currently employed as one of the main cellular factories for the expression of recombinant protein-based drugs. The establishment of high-producing cell lines typically begins with a heterogeneous starter population of cells, from which the highest producing cells are selected via empirical approaches. This approach is time consuming, and is likely to encounter natural upper limits imposed by the inherent biology of the cell lines in question. In an attempt to understand both the nature of the variability in populations of cells transfected with recombinant protein encoding DNA and the natural mechanisms of productivity limitation, we developed protocols for the detailed investigation of gene expression pathways in such cell lines. This novel approach was then applied to a set of clonal CHOK1 cell lines producing recombinant luciferase with varying productivities. Our results show that the initial limitation in these cell lines is at the transcriptional level, however in the highest producing cell line post-translational mechanisms affecting both protein turnover and protein folding become severely limiting. The implications for the development of strategies to engineer cells for enhanced recombinant protein production levels are discussed.

Production of recombinant proteins by sol–gel immobilized Escherichia coli

The aim of this work was to study the functionality and quantitative level of recombinant protein produced from bacteria immobilized and preserved in sol–gel matrices. Matrices prepared from two precursors, silicon dioxide and tetraethoxysilane, were studied. In previous works we analyzed the number of viable cells and level of recombinant protein production from cultures started with immobilized Escherichia coli, stored in sealed tubes at 4 and 20 °C. We observed that the amount of bacteria in silicon dioxide derived matrix conserved in the same order of magnitude as before immobilization, during 2 months, but those in an alkoxide derived matrix decrease until no viable cells were detected at both 4 and 20 °C after 42 days. In this work, immobilized bacteria were used as culture starter to produce recombinant proteins with a yield comparable with glycerol stocks. T-cell proliferation and gel filtration assays suggest that SAgs produced from cultures started with sol–gel immobilized bacteria retain their biological activity. Affinity assays using a resonant biosensor showed that Streptococcal Superantigen (SSA) has affinity for human Vβ5.2 produced from sol–gel immobilized bacteria with K D = 7.5 μM. These results contribute to the development of methods for microbial cells preservation under field conditions.

Genotyping of recombinant Pichia pastoris strains.

A simplified amplified-fragment length polymorphism (AFLP) method was used to genotype Pichia pastoris strains obtained by transformation of P. pastoris strain GS115 with a single integration vector. A total of 14 transformants and 3 control strains were analyzed, which generated 16 different band patterns. A clonal variation was obtained after the transformation process due to genetic differences generated during the transformation event of the host strain. Furthermore, the cluster analysis showed that the transformants with lesser genetic differences with respect to the P. pastoris host strain are the recombinant strains with the highest level of recombinant protein production.

Incorporation of pepstatin into culture media for reduction of protease activity in filtrates from Aspergillus niger NRRL-3

Aspergillus species have been used in different industrial applications including extracellular enzyme production and as potential hosts for recombinant heterologous proteins production because of their excellent growth and fermentation properties. However, recombinant protein production levels are typically low due to the impact of Aspergillus proteases in modifying and degrading the heterologous proteins, especially in the extracellular medium. The potential of using the protease inhibitor pepstatin in filamentous Aspergillus niger culture was investigated in this study. The majority of protease activity was released into the medium as biomass content decreases after maximum biomass concentration was attained and a low concentration of pepstatin effectively inhibited the extracellular protease activity to an extent of 80–90%. The effectiveness of pepstatin in inhibiting the majority of protease activity was further demonstrated in media supplemented with both non-peptide and peptide-containing nitrogen sources. Pepstatin addition had no effect on microbial growth. Dilution of the culture filtrate released inhibition, indicating that pepstatin did not have a notable effect on protease production and that inhibition was reversible.

Efficient preservation in a silicon oxide matrix of Escherichia coli, producer of recombinant proteins

The aim of this work was to study the use of silicon oxide matrices for the immobilization and preservation of recombinant-protein-producing bacteria. We immobilized Escherichia coli BL21 transformants containing different expression plasmids. One contained DNA coding for a T-cell receptor beta chain, which was expressed as inclusion bodies in the cytoplasm. The other two encoded bacterial superantigens Staphylococcal Enterotoxin G and Streptococcal Superantigen, which were expressed as soluble proteins in the periplasm. The properties of immobilization and storage stability in inorganic matrices prepared from two precursors, silicon dioxide and tetraethoxysilane, were studied. Immobilized E. coli was stored in sealed tubes at 4 and 20 degrees C and the number of viable cells and level of recombinant protein production were analyzed weekly. Different tests showed that the biochemical characteristics of immobilized E. coli remained intact. At both temperatures selected, we found that the number of bacteria in silicon dioxide-derived matrix was of the same order of magnitude (10(9) cfu ml(-1)) as before immobilization, for 2 months. After 2 weeks, cells immobilized in an alkoxide-derived matrix decreased to 10(4) cfu ml(-1) at 4 degrees C, and no viable cells were detected at 20 degrees C. We found that immobilized bacteria could be used as a starter to produce recombinant proteins with yields comparable to those obtained from glycerol stocks: 15 mg l(-1) for superantigens and 2 mg l(-1) for T-cell receptor beta chain. These results contribute to the development of methods for microbial cell preservation under field conditions.