High Yields Of Recombinant Protein Research Articles

A high-throughput, restriction-free cloning and screening strategy based on ccd B-gene replacement

BackgroundIn high-throughput demanding fields, such as biotechnology and structural biology, molecular cloning is an essential tool in obtaining high yields of recombinant protein. Here, we address recently developed restriction-free methods in cloning, and present a more cost-efficient protocol that has been optimized to improve both cloning and clone screening.ResultsIn our case study, three homologous β-lactamase genes were successfully cloned using these restriction-free protocols. To clone the genes, we chose a gene replacement strategy, where the recombinant genes contained overhangs that targeted a region of the expression vector including a cytotoxin-encoding ccdB-gene.ConclusionWe provide further evidence that gene replacement can be applied with high-throughput cloning protocols. Targeting a replacement of the ccdB-gene was found to be very successful for counterselection using these protocols. This eliminated the need for treatment with the restriction enzyme DpnI that has so far been the preferred clone selection approach. We thus present an optimized cloning protocol using a restriction-free ccdB-gene replacement strategy, which allows for parallel cloning at a high-throughput level.

Effects of temperature and glycerol and methanol‐feeding profiles on the production of recombinant galactose oxidase in Pichia pastoris

Optimization of protein production from methanol-induced Pichia pastoris cultures is necessary to ensure high productivity rates and high yields of recombinant proteins. We investigated the effects of temperature and different linear or exponential methanol-feeding rates on the production of recombinant Fusarium graminearum galactose oxidase (EC 1.1.3.9) in a P. pastoris Mut+ strain, under regulation of the AOX1 promoter. We found that low exponential methanol feeding led to 1.5-fold higher volumetric productivity compared to high exponential feeding rates. The duration of glycerol feeding did not affect the subsequent product yield, but longer glycerol feeding led to higher initial biomass concentration, which would reduce the oxygen demand and generate less heat during induction. A linear and a low exponential feeding profile led to productivities in the same range, but the latter was characterized by intense fluctuations in the titers of galactose oxidase and total protein. An exponential feeding profile that has been adapted to the apparent biomass concentration results in more stable cultures, but the concentration of recombinant protein is in the same range as when constant methanol feeding is employed. © 2014 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 30:728–735, 2014

Differential affects of low glucose on the macroheterogeneity and microheterogeneity of glycosylation in CHO-EG2 camelid monoclonal antibodies

Background The demand for high yield recombinant protein production systems has focused industry on culture media and feed strategies that optimize productivity, yet maintain product quality attributes such as glycosylation. Minimizing media components such as glucose, reduces the production of lactate, but may also affect glycosylation. The first steps in the glycosylation pathway involve the synthesis of lipid-linked oligosaccharides (LLOs). Glycan macroheterogeneity is introduced by variation in site-specific glycosylation with the transfer of the oligosaccharide to the protein. Further modification of the oligosaccharide can occur through processing reactions, where some sugars are removed and additional sugars added. This produces microheterogeneity of the glycan pool. Both macroheterogeneity and microheterogeneity may be affected by fermentation conditions. The objective of this study has been to investigate the effect of variable concentrations of glucose on the glycosylation patterns of a camelid monoclonal antibody produced in Chinese hamster ovary (CHO) cells and to further evaluate their effect on components of the N-glycosylation pathway.

Recent advances on host plants and expression cassettes' structure and function in plant molecular pharming

Plant molecular pharming is a promising system to produce important recombinant proteins such as therapeutic antibodies, pharmaceuticals, enzymes, growth factors, and vaccines. The system provides an interesting alternative method to the direct extraction of proteins from inappropriate source material while offering the possibility to overcome problems related to product safety and source availability. Multiple factors including plant hosts, genes of interest, expression vector cassettes, and extraction and purification techniques play important roles in the plant molecular pharming. Plant species, as a biosynthesis platform, are a crucial factor in achieving high yields of recombinant protein in plant. The choice of recombinant gene and its expression strategy is also of great importance in ensuring a high amount of the recombinant proteins. Many studies have been conducted to improve expression, accumulation, and purification of the recombinant protein from molecular pharming systems. Re-engineered vectors and expression cassettes are also pivotal tools in enhancing gene expression at the transcription and translation level, and increasing protein accumulation, stability, retention and targeting of specific organelles. In this review, we report recent advances and strategies of plant molecular pharming while focusing on the choice of plant hosts and the role of some molecular pharming elements and approaches: promoters, codon optimization, signal sequences, and peptides used for upstream design, purification and downstream processing.

The pEAQ vector series: the easy and quick way to produce recombinant proteins in plants

The pEAQ vectors are a series of plasmids designed to allow easy and quick production of recombinant proteins in plants. Their main feature is the use of the Cowpea Mosaic Virus hypertranslational "CPMV-HT" expression system, which provides high yields of recombinant protein through extremely high translational efficiency without the need for viral replication. Since their creation, the pEAQ vectors have been used to produce a wide variety of proteins in plants. Viral proteins and Virus-Like Particles (VLPs) have been of particular interest, but other types of proteins including active enzymes have also been expressed. While the pEAQ vectors have mostly been used in a transient expression context, through agroinfiltration of leaves, they have also been shown to be suitable for the production of stably transformed lines of both cell cultures and whole plants. This paper looks back on the genesis of the pEAQ vectors and reviews their use so far.

Recombinant expression of the coat protein of Botrytis virus X and development of an immunofluorescence detection method to study its intracellular distribution in Botrytis cinerea

Botrytis cinerea is infected by many mycoviruses with varying phenotypical effects on the fungal host, including Botrytis virus X (BVX), a mycovirus that has been found in several B. cinerea isolates worldwide with no obvious effects on growth. Here we present results from serological and immunofluorescence microscopy (IFM) studies using antiserum raised against the coat protein of BVX expressed in Escherichia coli fused to maltose-binding protein. Due to the high yield of recombinant protein it was possible to raise antibodies that recognized BVX particles. An indirect ELISA, using BVX antibodies, detected BVX in partially purified virus preparations from fungal isolates containing BVX alone and in mixed infection with Botrytis virus F. The BVX antiserum also proved suitable for IFM studies. Intensely fluorescing spots (presumed to be virus aggregates) were found to be localized in hyphal cell compartments and spores of natural and experimentally infected B. cinerea isolates using IFM. Immunofluorescently labelled sections through fungal tissue, as well as fixed mycelia grown on glass slides, showed aggregations of virions closely associated with fungal cell membranes and walls, next to septal pores, and in hyphal tips. Also, calcofluor white staining of mature cell walls of virus-transfected Botrytis clones revealed numerous cell wall areas with increased amounts of chitin/glycoproteins. Our results indicate that some BVX aggregates are closely associated with the fungal cell wall and raise the question of whether mycoviruses may be able to move through the wall and therefore not be totally dependent on intracellular routes of transmission.

The development of a high-yield recombinant protein bioreactor through RNAi induced knockdown of ATP/ADP transporter in Solanum tuberosum

There is an increased need for high-yield protein production platforms to meet growing demand. Tuber-based production in Solanum tuberosum offers several advantages, including high biomass yield, although protein concentration is typically low. In this work, we investigated the question whether minor interruption of starch biosynthesis can have a positive effect on tuber protein content and/or tuber biomass, as previous work suggested that partial obstruction of starch synthesis had variable effects on tuber yield. To this end, we used a RNAi approach to knock down ATP/ADP transporter and obtained a large number of transgenic lines for screening of lines with improved tuber protein content and/or tuber biomass. The initial screening was based on tuber biomass because of its relative simplicity. We identified a line, riAATP1-10, with minor (less than 15%) reduction in starch, that had a nearly 30% increase in biomass compared to wild-type, producing both more and larger tubers with altered morphological features compared to wild-type. riAATP1-10 tubers have a higher concentration of soluble protein compared to wild-type tubers, with nearly 50% more soluble protein. We assessed the suitability of this line as a new bioreactor by expressing a human scFv, reaching over 0.5% of total soluble protein, a 2-fold increase over the highest accumulating line in a wild-type background. Together with increased biomass and increased levels in total protein content, foreign protein expression in riAATP1-10 line would translate into a nearly 4-fold increase in recombinant protein yield per plant. Our results indicate that riAATP1-10 line provides an improved expression system for production of foreign proteins.

Optimization of prokaryotic expression condition and purification of anti-cancer protein NOR1 in E.coli

To optimize the induction condition of human NOR1 gene expression in E.coli. and purify NOR1 recombinant proteins. A full-length cDNA of human NOR1 was inserted into the corresponding region of pET28b expression vector to yield recombinant prokaryotic expression vector pET28b-NOR1. The prokaryotic expression vector pET28b-NOR1 was introduced into the bacterial host E.coli Rosettablue(DE3). Recombinant NOR1 protein was induced at different conditions. Induction condition was optimized to obtain high yield of recombinant protein. At last, the recombinant NOR1 protein was purified by Ni-IDE chromatography resin. Recombinant NOR1 protein was induced by IPTG in a dose-dependent manner. Increase of kanamycin concentration and induction temperature resulted in high yield of recombinant protein. The most recombinant protein was found in inclusion bodies. The recombinant His-NOR1 protein was purified with Ni-IDE chromatography resin under denature condition. IPTG, kanamycin concentration and temperature can affect the expression of recombinant NOR1 protein in pET28b system. High yield of recombinant NOR1 protein is achieved by inducing 1 mmol/L IPTG and 200 μg/mL kanamycin at 37 degree. Recombinant His-NOR1 protein with high purity is purified.

Developing a scalable model of recombinant protein yield from Pichia pastoris: the influence of culture conditions, biomass and induction regime

BackgroundThe optimisation and scale-up of process conditions leading to high yields of recombinant proteins is an enduring bottleneck in the post-genomic sciences. Typical experiments rely on varying selected parameters through repeated rounds of trial-and-error optimisation. To rationalise this, several groups have recently adopted the 'design of experiments' (DoE) approach frequently used in industry. Studies have focused on parameters such as medium composition, nutrient feed rates and induction of expression in shake flasks or bioreactors, as well as oxygen transfer rates in micro-well plates. In this study we wanted to generate a predictive model that described small-scale screens and to test its scalability to bioreactors.ResultsHere we demonstrate how the use of a DoE approach in a multi-well mini-bioreactor permitted the rapid establishment of high yielding production phase conditions that could be transferred to a 7 L bioreactor. Using green fluorescent protein secreted from Pichia pastoris, we derived a predictive model of protein yield as a function of the three most commonly-varied process parameters: temperature, pH and the percentage of dissolved oxygen in the culture medium. Importantly, when yield was normalised to culture volume and density, the model was scalable from mL to L working volumes. By increasing pre-induction biomass accumulation, model-predicted yields were further improved. Yield improvement was most significant, however, on varying the fed-batch induction regime to minimise methanol accumulation so that the productivity of the culture increased throughout the whole induction period. These findings suggest the importance of matching the rate of protein production with the host metabolism.ConclusionWe demonstrate how a rational, stepwise approach to recombinant protein production screens can reduce process development time.

Practical protocols for production of very high yields of recombinant proteins using Escherichia coli

The gram-negative bacterium Escherichia coli offers a mean for rapid, high yield, and economical production of recombinant proteins. However, high-level production of functional eukaryotic proteins in E. coli may not be a routine matter, sometimes it is quite challenging. Techniques to optimize heterologous protein overproduction in E. coli have been explored for host strain selection, plasmid copy numbers, promoter selection, mRNA stability, and codon usage, significantly enhancing the yields of the foreign eukaryotic proteins. We have been working on optimizations of bacterial expression conditions and media with a focus on achieving very high cell density for high-level production of eukaryotic proteins. Two high-cell-density bacterial expression methods have been explored, including an autoinduction introduced by Studier (Protein Expr Purif 2005;41:207-234) recently and a high-cell-density IPTG-induction method described in this study, to achieve a cell-density OD(600) of 10-20 in the normal laboratory setting using a regular incubator shaker. Several practical protocols have been implemented with these high-cell-density expression methods to ensure a very high yield of recombinant protein production. With our methods and protocols, we routinely obtain 14-25 mg of NMR triple-labeled proteins and 17-34 mg of unlabeled proteins from a 50-mL cell culture for all seven proteins we tested. Such a high protein yield used the same DNA constructs, bacterial strains, and a regular incubator shaker and no fermentor is necessary. More importantly, these methods allow us to consistently obtain such a high yield of recombinant proteins using E. coli expression.

Altering the ribosomal subunit ratio in yeast maximizes recombinant protein yield

BackgroundThe production of high yields of recombinant proteins is an enduring bottleneck in the post-genomic sciences that has yet to be addressed in a truly rational manner. Typically eukaryotic protein production experiments have relied on varying expression construct cassettes such as promoters and tags, or culture process parameters such as pH, temperature and aeration to enhance yields. These approaches require repeated rounds of trial-and-error optimization and cannot provide a mechanistic insight into the biology of recombinant protein production. We published an early transcriptome analysis that identified genes implicated in successful membrane protein production experiments in yeast. While there has been a subsequent explosion in such analyses in a range of production organisms, no one has yet exploited the genes identified. The aim of this study was to use the results of our previous comparative transcriptome analysis to engineer improved yeast strains and thereby gain an understanding of the mechanisms involved in high-yielding protein production hosts.ResultsWe show that tuning BMS1 transcript levels in a doxycycline-dependent manner resulted in optimized yields of functional membrane and soluble protein targets. Online flow microcalorimetry demonstrated that there had been a substantial metabolic change to cells cultured under high-yielding conditions, and in particular that high yielding cells were more metabolically efficient. Polysome profiling showed that the key molecular event contributing to this metabolically efficient, high-yielding phenotype is a perturbation of the ratio of 60S to 40S ribosomal subunits from approximately 1:1 to 2:1, and correspondingly of 25S:18S ratios from 2:1 to 3:1. This result is consistent with the role of the gene product of BMS1 in ribosome biogenesis.ConclusionThis work demonstrates the power of a rational approach to recombinant protein production by using the results of transcriptome analysis to engineer improved strains, thereby revealing the underlying biological events involved.

Characterization of gene localization and accessibility in DHFR‐amplified CHO cells

Efficient transcription is critical for high yields of recombinant proteins by mammalian cells. We previously reported that dihydrofolate reductase (DHFR)-mediated gene amplification can augment transcriptional rates as well as increasing gene copy numbers in Chinese hamster ovary (CHO) cells.1 In an attempt to elucidate the mechanisms involved, we have employed several approaches to identify the epigenetic differences between cell clones with varying transcriptional rates. Transgene placement and accessibility varies between unrelated parental cell clones with differential transcriptional rates. However, we did not observe any apparent epigenetic differences between parental clones and their amplified progeny, indicating undiscovered regulatory mechanisms are responsible for the augmentation of transcriptional rates upon DHFR-mediated amplification.

A Baculovirus Expression Vector System for Simultaneous Protein Expression in Insect and Mammalian Cells

Since the number of potential drug targets identified has significantly increased in the past decade, rapid expression of recombinant proteins in sufficient amounts for structure determination and modern drug discovery is one of the major challenges in pharmaceutical research. As a result of its capacity for insertion of large DNA fragments, its high yield of recombinant protein and its high probability of success compared to protein expression in Escherichia coli, the baculovirus expression vector system (BEVS) is used routinely to produce recombinant proteins in the milligram scale. For some targets, however, expression of the recombinant protein with the BEVS in insect cells fails and mammalian expression systems have to be used to achieve proper post-translational processing of the nascent polypeptide. We now introduce a modified BEVS as a very useful tool for simultaneously testing the expression of target proteins in both insect and mammalian cells by using baculovirus infection of both host systems. The expression yields in insect cells are comparable to those obtained with state-of-the-art baculovirus vectors, such as the Bac-to-Bac system. Using the same virus, we can transduce mammalian cells to quickly assess target gene expression feasibility and optimize expression conditions, eliminating additional cloning steps into mammalian expression vectors. This reduces time and effort for finding appropriate expression conditions in various hosts.

Posttransformational vector amplification in the yeastPichia pastoris

Generating a high yield of recombinant protein is a major goal when expressing a foreign gene in any expression system. In the methylotrophic yeast Pichia pastoris, a common means of achieving this end is to select for transformants containing multiple integrated copies of an expression vector by plating them on high levels of a selectable marker drug followed by screening for rare colonies with multiple copies. We describe a more convenient method to select for such clones. Using Zeocin-resistance-based vectors, we demonstrate that strains transformed with only one or a few vector copies can, long after transformation, be subjected to further selection at high levels of drug. This resulted in the frequent selection of clones containing increased copy numbers of the vector. This posttransformational vector amplification (PTVA) process resulted in strains containing multiple head-to-tail copies of the entire vector integrated at a single locus in the genome. Of our PTVA selected clones, 40% showed a three- to fivefold increase in vector copy number. So-called 'jackpot' clones with >10 copies of the expression vector represented 5-6% of selected clones and had a proportional increase in recombinant protein.

Serum‐free transient protein production system based on adenoviral vector and PER.C6 technology: High yield and preserved bioactivity

Stable E1 transformed cells, like PER.C6, are able to grow at scale and to high cell densities. E1‐deleted adenoviruses replicate to high titer in PER.C6 cells whereas subsequent deletion of E2A from the vector results in absence of replication in PER.C6 cells and drastically lowers the expression of adenovirus proteins in such cells. We therefore considered the use of an ΔE1/ΔE2 type 5 vector (Ad5) to deliver genes to PER.C6 cells growing in suspension with the aim to achieve high protein yield. To evaluate the utility of this system we constructed ΔE1/ΔE2 vector carrying different classes of protein, that is, the gene coding for spike protein derived from the Coronavirus causing severe acute respiratory syndrome (SARS‐CoV), a gene coding for the SARS‐CoV receptor or the genes coding for an antibody shown to bind and neutralize SARS‐CoV (SARS‐AB). The ΔE1/ΔE2A‐vector backbones were rescued on a PER.C6 cell line engineered to constitutively over express the Ad5 E2A protein. Exposure of PER.C6 cells to low amounts (30 vp/cell) of ΔE1/ΔE2 vectors resulted in highly efficient (>80%) transduction of PER.C6 cells growing in suspension. The efficient cell transduction resulted in high protein yield (up to 60 picogram/cell/day) in a 4 day batch production protocol. FACS and ELISA assays demonstrated the biological activity of the transiently produced proteins. We therefore conclude that ΔE1/ΔE2 vectors in combination with the PER.C6 technology may provide a viable answer to the increasing demand for high quality, high yield recombinant protein. Biotechnol. Bioeng. 2008;100: 273–283. © 2007 Wiley Periodicals, Inc.

Chlamydomonas reinhardtii chloroplasts as protein factories

Protein-based therapeutics are the fastest growing sector of drug development, mainly because of the high sensitivity and specificity of these molecules. Their high specificity leads to few side effects and excellent success rates in drug development. However, the inherent complexity of these molecules restricts their synthesis to living cells, making recombinant proteins expensive to produce. In addition to therapeutic uses, recombinant proteins also have a variety of industrial applications and are important research reagents. Eukaryotic algae offer the potential to produce high yields of recombinant proteins more rapidly and at much lower cost than traditional cell culture. Additionally, transgenic algae can be grown in complete containment, reducing any risk of environmental contamination. This system might also be used for the oral delivery of therapeutic proteins, as green algae are edible and do not contain endotoxins or human viral or prion contaminants.

A regulatory acceptable alternative to E. coli : high yield recombinant protein production using the Lactococcus lactis P170 expression system combined with "Reverse electro enhanced dialysis" (REED) for lactate control

view on host physiology The organisers would like to thank Novozymes Delta Ltd who generously supported the meeting. Meeting

Prokaryotical expression of structural and non-structural proteins of hepatitis G virus.

To study the epitope distribution of hepatitis G virus (HGV) and to seek for the potential recombinant antigens for the development of HGV diagnostic reagents. Fourteen clones encompassing HGV gene fragments from core to NS3 and NS5 were constructed using prokaryotic expression vector pRSET and (or) pGEX, and expressed in E.coli. Western blotting and ELISA were used to detect the immunoreactivity of these recombinant proteins. One clone with HGV fragment from core to E1 (G1), one from E2 (G31), three from NS3 (G6, G61, G7), one from NS5B (G821) and one chimeric fragment from NS3 and NS5B (G61-821) could be expressed well and showed obvious immunoreactivity by Western blotting. One clone with HGV framment from NS5B (G82) was also well expressed, but could not show immunoreactivity by Western blotting. No obvious expression was found in the other six clones. All the expressed recombinant proteins were in inclusion body form, except the protein G61 which could be expressed in soluble form. Further purified recombinant proteins G1, G31, G61, G821 and G61-821 were detected in indirected ELISA as coating antigen respectively. Only recombinant G1 could still show immunoreactivity, and the other four recombinant proteins failed to react to the HGV antibody positive sera. Western blotting results indicated that the immunoactivity of these four recombinant proteins were lost during purification. Core to E1, E2, NS3 and NS5 fragment of HGV contain antigenic epitopes, which could be produced in prokaryotically expressed recombinant proteins. A high-yield recombinant protein (G1) located in HGV core to E1 could remain its epitope after purification, which showed the potential that G1 could be used as a coating antigen to develop an ELISA kit for HGV specific antibody diagnosis.

Design of high-throughput methods of protein production for structural biology

I greatly appreciate interacting with Peter Schultz, Scott Lesley and Marc Nasoff at the Genomics Institute for the Novartis Research Foundation and Mark Knuth and Ron Swanson at Syrrx, Inc., where much of this work is currently being pursued. I also thank Jim Graziano, Chris Lee, Mike Hanson and Marianne Patch for helpful suggestions on this manuscript.

An early step of glycosylphosphatidyl-inositol anchor biosynthesis is abolished in lepidopteran insect cells following baculovirus infection.

The expression of recombinant proteins in their native state has become a prerequisite for a variety of functional and structural studies, as well as vaccine development. Many biochemical properties and functions of proteins are dependent on or reside in posttranslational modifications, such as glycosylation. The baculovirus system has increasingly become the system of choice due to it capabilities of performing posttranslational modifications and usually high yields of recombinant proteins. The Toxoplasma gondii surface antigen SAG1 was used as a model for a glycosylphosphatidyl-inositol (GPI)-anchored protein and expressed in insect cells using the baculovirus system. We show that the T. gondii SAG1 surface antigen expressed in this system was not modified by a GPI-anchor. In vitro and in vivo studies demonstrate that uninfected insect cells are able to produce GPI-precursors and to transfer a mature GPI-anchor to nascent proteins. These cells however are not capable to produce GPI-precursors following infection. We also show that the biosynthesis of the early GPI intermediate GlcNH(2)-PI is blocked in baculovirus-infected H5 cells, thus preventing the subsequent mannosylation steps for the synthesis of the conserved GPI-core-glycan. We therefore conclude that the baculovirus system is not appropriate for the expression of GPI-anchored proteins.