Optimization of elastin‐like polypeptide fusions for expression and purification of recombinant proteins in plants

The demand for recombinant proteins for medical and industrial use is expanding rapidly and plants are now recognized as an efficient, inexpensive means of production. Although the accumulation of recombinant proteins in transgenic plants can be low, we have previously demonstrated that fusions with an elastin‐like polypeptide (ELP) tag can significantly enhance the production yield of a range of different recombinant proteins in plant leaves. ELPs are biopolymers with a repeating pentapeptide sequence (VGVPG)_n that are valuable for bioseparation, acting as thermally responsive tags for the non‐chromatographic purification of recombinant proteins. To determine the optimal ELP size for the accumulation of recombinant proteins and their subsequent purification, various ELP tags were fused to green fluorescent protein, interleukin‐10, erythropoietin and a single chain antibody fragment and then transiently expressed in tobacco leaves. Our results indicated that ELP tags with 30 pentapeptide repeats provided the best compromise between the positive effects of small ELP tags (n = 5–40) on recombinant protein accumulation and the beneficial effects of larger ELP tags (n = 80–160) on recombinant protein recovery during inverse transition cycling (ITC) purification. In addition, the C‐terminal orientation of ELP fusion tags produced higher levels of target proteins, relative to N‐terminal ELP fusions. Importantly, the ELP tags had no adverse effect on the receptor binding affinity of erythropoietin, demonstrating the inert nature of these tags. The use of ELP fusion tags provides an approach for enhancing the production of recombinant proteins in plants, while simultaneously assisting in their purification. Biotechnol. Bioeng. 2009;103: 562–573. © 2009 Wiley Periodicals, Inc.     

Production of a tumour‐targeting antibody with a human‐compatible glycosylation profile in N. benthamiana hairy root cultures

Hairy root (HR) cultures derived from Agrobacterium rhizogenes transformation of plant tissues are an advantageous biotechnological manufacturing platform due to the accumulation of recombinant proteins in an otherwise largely protein free culture medium. In this context, HRs descending from transgenic Nicotiana tabacum plants were successfully used for the production of several functional mAbs with plant‐type glycans. Here, we expressed the tumor‐targeting monoclonal antibody mAb H10 in HRs obtained either by infecting a transgenic N. tabacum line expressing H10 with A. rhizogenes or a glyco‐engineered N. benthamiana line (ΔXTFT) with recombinant A. rhizogenes carrying mAb H10 heavy and light chain cDNAs. Selected HR clones derived from both plants accumulated mAb H10 in the culture medium with similar yields (2–3 mg/L). N‐glycosylation profiles of antibodies purified from HR supernatant revealed the presence of plant‐typical complex structures for N. tabacum‐derived mAb H10 and of GnGn structures lacking xylose and fucose for the ΔXTFT‐derived counterpart. Both antibody glyco‐formats exhibited comparable antigen binding activities. Collectively, these data demonstrate that the co‐infection of ΔXTFT Nicotiana benthamiana with recombinant A. rhizogenes is an efficient procedure for the generation of stable HR cultures expressing the tumor‐targeting mAb H10 with a human‐compatible glycosylation profile, thus representing an important step towards the exploitation of root cultures for the production of ‘next generation’ human therapeutic antibodies.     

Growth hormone and gene expression of in vitro‐matured rhesus macaque oocytes

Growth hormone (GH) in rhesus macaque in vitro oocyte maturation (IVM) has been shown to increase cumulus expansion and development of embryos to the 9–16 cell stage in response to 100 ng/ml recombinant human GH (r‐hGH) supplementation during IVM. Although developmental endpoints for metaphase II (MII) oocytes and embryos are limited in the macaque, gene expression analysis can provide a mechanism to explore GH action on IVM. In addition, gene expression analysis may allow molecular events associated with improved cytoplasmic maturation to be detected. In this study, gene expression of specific mRNAs in MII oocytes and cumulus cells that have or have not been exposed to r‐hGH during IVM was compared. In addition, mRNA expression was compared between in vitro and in vivo‐matured metaphase II (MII) oocytes and germinal vesicle (GV)‐stage oocytes. Only 2 of 17 genes, insulin‐like growth factor 2 (IGF2) and steroidogenic acute regulator (STAR), showed increased mRNA expression in MII oocytes from the 100 ng/ml r‐hGH treatment group compared with other IVM treatment groups, implicating insulin‐like growth factor (IGF) and steroidogenesis pathways in the oocyte response to GH. The importance of IGF2 is notable, as expression of IGF1 was not detected in macaque GV‐stage or MII oocytes or cumulus cells. Mol. Reprod. Dev. 77: 353–362, 2010. © 2009 Wiley‐Liss, Inc.     

MALDI‐TOF characterization of hGH1 produced by hairy root cultures of Brassica oleracea var. italica grown in an airlift with mesh bioreactor

Expression systems based on plant cells, tissue, and organ cultures have been investigated as an alternative for production of human therapeutic proteins in bioreactors. In this work, hairy root cultures of Brassica oleracea var. italica (broccoli) were established in an airlift with mesh bioreactor to produce isoform 1 of the human growth hormone (hGH1) as a model therapeutic protein. The hGH1 cDNA was cloned into the pCAMBIA1105.1 binary vector to induce hairy roots in hypocotyls of broccoli plantlets via Agrobacterium rhizogenes. Most of the infected plantlets (90%) developed hairy roots when inoculated before the appearance of true leaves, and keeping the emerging roots attached to hypocotyl explants during transfer to solid Schenk and Hildebrandt medium. The incorporation of the cDNA into the hairy root genome was confirmed by PCR amplification from genomic DNA. The expression and structure of the transgenic hGH1 was assessed by ELISA, western blot, and MALDITOF‐MS analysis of the purified protein extracted from the biomass of hairy roots cultivated in bioreactor for 24 days. Production of hGH1 was 5.1 ± 0.42 µg/g dry weight (DW) for flask cultures, and 7.8 ± 0.3 µg/g DW for bioreactor, with productivity of 0.68 ± 0.05 and 1.5 ± 0.06 µg/g DW*days, respectively, indicating that the production of hGH1 was not affected by the growth rate, but might be affected by the culture system. These results demonstrate that hairy root cultures of broccoli have potential as an alternative expression system for production of hGH1, and might also be useful for production of other therapeutic proteins. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:161–171, 2014     

Optimization of the bioprocessing conditions for scale‐up of transient production of a heterologous protein in plants using a chemically inducible viral amplicon expression system

Use of transient expression for the rapid, large‐scale production of recombinant proteins in plants requires optimization of existing methods to facilitate scale‐up of the process. We have demonstrated that the techniques used for agroinfiltration and induction greatly impact transient production levels of heterologous protein. A Cucumber mosaic virus inducible viral amplicon (CMViva) expression system was used to transiently produce recombinant alpha‐1‐antitrypsin (rAAT) by co‐infiltrating harvested Nicotiana benthamiana leaves with two Agrobacterium tumefaciens strains, one containing the CMViva expression cassette carrying the AAT gene and the other containing a binary vector carrying the gene silencing suppressor p19. Harvested leaves were both infiltrated and induced by either pressure or vacuum infiltration. Using the vacuum technique for both processes, maximum levels of functional and total rAAT were elevated by (190 ± 8.7)% and (290 ± 7.5)%, respectively, over levels achieved when using the pressure technique for both processes. The bioprocessing conditions for vacuum infiltration and induction were optimized and resulted in maximum rAAT production when using an A. tumefaciens concentration at OD₆₀₀ of 0.5 and a 0.25‐min vacuum infiltration, and multiple 1‐min vacuum inductions further increased production 25% and resulted in maximum levels of functional and total rAAT at (2.6 ± 0.09)% and (4.1 ± 0.29)% of the total soluble protein, respectively, or (90 ± 1.7) and (140 ± 10) mg per kg fresh weight leaf tissue at 6 days post‐induction. Use of harvested plant tissue with vacuum infiltration and induction demonstrates a bioprocessing route that is fully amenable to scale‐up. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Plant‐derived recombinant human serum transferrin demonstrates multiple functions

Human serum transferrin (hTf) is the major iron‐binding protein in human plasma, having a vital role in iron transport. Additionally, hTf has many other uses including antimicrobial functions and growth factor effects on mammalian cell proliferation and differentiation. The multitask nature of hTf makes it highly valuable for different therapeutic and commercial applications. However, the success of hTf in these applications is critically dependent on the availability of high‐quality hTf in large amounts. In this study, we have developed plants as a novel platform for the production of recombinant (r)hTf. We show here that transgenic plants are an efficient system for rhTf production, with a maximum accumulation of 0.25% total soluble protein (TSP) (or up to 33.5 μg/g fresh leaf weight). Furthermore, plant‐derived rhTf retains many of the biological activities synonymous with native hTf. In particular, rhTf reversibly binds iron in vitro, exhibits bacteriostatic activity, supports cell proliferation in serum‐free medium and can be internalized into mammalian cells in vitro. The success of this study validates the future application of plant rhTf in a variety of fields. Of particular interest is the use of plant rhTf as a novel carrier for cell‐specific or oral delivery of protein/peptide drugs for the treatment of human diseases such as diabetes. To demonstrate this hypothesis, we have additionally expressed an hTf fusion protein containing glucagon‐like peptide 1 (GLP‐1) or its derivative in plants. Here, we show that plant‐derived hTf‐GLP‐1 fusion proteins retain the ability to be internalized by mammalian cells when added to culture medium in vitro.     

Escherichia coli “TatExpress” strains super‐secrete human growth hormone into the bacterial periplasm by the Tat pathway

Numerous high‐value proteins are secreted into the Escherichia coli periplasm by the General Secretory (Sec) pathway, but Sec‐based production chassis cannot handle many potential target proteins. The Tat pathway offers a promising alternative because it transports fully folded proteins; however, yields have been too low for commercial use. To facilitate Tat export, we have engineered the TatExpress series of super‐secreting strains by introducing the strong inducible bacterial promoter, ptac , upstream of the chromosomal tatABCD operon, to drive its expression in E. coli strains commonly used by industry (e.g., W3110 and BL21). This modification significantly improves the Tat‐dependent secretion of human growth hormone (hGH) into the bacterial periplasm, to the extent that secreted hGH is the dominant periplasmic protein after only 1 hr induction. TatExpress strains accumulate in excess of 30 mg L^?1 periplasmic recombinant hGH, even in shake flask cultures. A second target protein, an scFv, is also shown to be exported at much higher rates in TatExpress strains.

     

Plant cell packs: a scalable platform for recombinant protein production and metabolic engineering

Industrial plant biotechnology applications include the production of sustainable fuels, complex metabolites and recombinant proteins, but process development can be impaired by a lack of reliable and scalable screening methods. Here, we describe a rapid and versatile expression system which involves the infusion of Agrobacterium tumefaciens into three‐dimensional, porous plant cell aggregates deprived of cultivation medium, which we have termed plant cell packs (PCPs). This approach is compatible with different plant species such as Nicotiana tabacum BY2, Nicotiana benthamiana or Daucus carota and 10‐times more effective than transient expression in liquid plant cell culture. We found that the expression of several proteins was similar in PCPs and intact plants, for example, 47 and 55 mg/kg for antibody 2G12 expressed in BY2 PCPs and N. tabacum plants respectively. Additionally, the expression of specific enzymes can either increase the content of natural plant metabolites or be used to synthesize novel small molecules in the PCPs. The PCP method is currently scalable from a microtiter plate format suitable for high‐throughput screening to 150‐mL columns suitable for initial product preparation. It therefore combined the speed of transient expression in plants with the throughput of microbial screening systems. Plant cell packs therefore provide a convenient new platform for synthetic biology approaches, metabolic engineering and conventional recombinant protein expression techniques that require the multiplex analysis of several dozen up to hundreds of constructs for efficient product and process development.     

Expression system for recombinant human growth hormone production from Bacillus subtilis

We demonstrate for the first time, an expression system mimicking serine alkaline protease synthesis and secretion, producing native form of human growth hormone (hGH) from Bacillus subtilis. A hybrid‐gene of two DNA fragments, i.e., signal (pre‐) DNA sequence of B. licheniformis serine alkaline protease gene (subC) and cDNA encoding hGH, were cloned into pMK4 and expressed under deg‐promoter in B. subtilis. Recombinant‐hGH (rhGH) produced by B. subtilis carrying pMK4::pre(subC)::hGH was secreted. N‐terminal sequence and mass spectrometry analyses of rhGH confirm the mature hGH sequence, and indicate that the signal peptide was properly processed by B. subtilis signal‐peptidase. The highest rhGH concentration was obtained at t = 32 h as C_rhGH = 70 mg L^?1 with a product yield on substrate Y_rhGH/S = 9 g kg^?1, in a glucose based defined medium. Fermentation characteristics and influence of hGH gene on the rhGH production were investigated by comparing B. subtilis carrying pMK4::pre(subC)::hGH with that of carrying merely pMK4. Excreted organic‐acid concentrations were higher by B. subtilis carrying pMK4::pre(subC)::hGH, whereas excreted amino‐acid concentrations were higher by B. subtilis carrying pMK4. The approach developed is expected to be applicable to the design of expression systems for heterologous protein production from Bacillus species. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Protein production from recombinant protein bodies

In this study, we describe a process for protein expression and purification from plants and insect cells based on the accumulation of recombinant proteins in protein bodies. This technology is using Zera^®, which sequence has the capacity to trigger in vivo the formation of dense, non-secretory storage protein body-like organelles derived from the endoplasmic reticulum (ER). With this method, recombinant human growth hormone (hGH) was expressed and purified from protein bodies accumulated in plants (Nicotiana benthamiana) and in insect cells (Spodoptera frugiperda). We found that recombinant Zera-hGH are stored in large quantity inside those proteins bodies and can be easily recovered during a one-step process from plant and insect cell biomass. After solubilization of recombinant protein bodies and cleavage of Zera tag from the fusion protein, active hGH was finally purified by a single chromatography step. These results indicate that recombinant proteins derived from Zera-fusion could provide both an efficient protein production system and eased purification downstream process.     

Induction of a dwarf phenotype with IBH1 may enable increased production of plant‐made pharmaceuticals in plant factory conditions

Year‐round production in a contained, environmentally controlled ‘plant factory’ may provide a cost‐effective method to produce pharmaceuticals and other high‐value products. However, cost‐effective production may require substantial modification of the host plant phenotype; for example, using dwarf plants can enable the growth of more plants in a given volume by allowing more plants per shelf and enabling more shelves to be stacked vertically. We show here that the expression of the chimeric repressor for Arabidopsis AtIBH1 (P35S:AtIBH1SRDX) in transgenic tobacco plants (Nicotiana tabacum) induces a dwarf phenotype, with reduced cell size. We estimate that, in a given volume of cultivation space, we can grow five times more AtIBH1SRDX plants than wild‐type plants. Although, the AtIBH1SRDX plants also showed reduced biomass compared with wild‐type plants, they produced about four times more biomass per unit of cultivation volume. To test whether the dwarf phenotype affects the production of recombinant proteins, we expressed the genes for anti‐hepatitis B virus antibodies (anti‐HBs) in tobacco plants and found that the production of anti‐HBs per unit fresh weight did not significantly differ between wild‐type and AtIBH1SRDX plants. These data indicate that P35S:AtIBH1SRDX plants produced about fourfold more antibody per unit of cultivation volume, compared with wild type. Our results indicate that AtIBH1SRDX provides a useful tool for the modification of plant phenotype for cost‐effective production of high‐value products by stably transformed plants in plant factory conditions.     

用Bac-to-Bac杆状病毒系统表达人生长激素

利用Bac to Bac杆状病毒载体表达系统将人生长激素 (humangrowthhormone ,hGH)基因cDNA克隆至转移载体pFastBac1中 ,得到pFastBac hGH ,再将其转化进入含穿梭载体Bacmid的受体菌DH10Bac中 ,发生转座作用 ,得到含hGH基因的重组穿梭载体rBacmid hGH .纯化DNA ,直接转染培养的昆虫细胞Sf9,得到重组病毒rAcV Bac hGH .经酶切PCR及Southern杂交鉴定 ,hGH基因正确地插入病毒基因组的多角体蛋白基因启动子下 ,SDS PAGE测得产物蛋白分子量为 2 2kD左右 .用免疫化学发光法测得转染上清中hGH表达水平可达 18μg ml ,与用传统的BEVS表达hGH相比 ,转染上清中hGH表达水平提高 4 0 0倍以上     

Development of Beet necrotic yellow vein virus‐based vectors for multiple‐gene expression and guide RNA delivery in plant genome editing

Many plant viruses with monopartite or bipartite genomes have been developed as efficient expression vectors of foreign recombinant proteins. Nonetheless, due to lack of multiple insertion sites in these plant viruses, it is still a big challenge to simultaneously express multiple foreign proteins in single cells. The genome of Beet necrotic yellow vein virus (BNYVV) offers an attractive system for expression of multiple foreign proteins owning to a multipartite genome composed of five positive‐stranded RNAs. Here, we have established a BNYVV full‐length infectious cDNA clone under the control of the Cauliflower mosaic virus 35S promoter. We further developed a set of BNYVV‐based vectors that permit efficient expression of four recombinant proteins, including some large proteins with lengths up to 880 amino acids in the model plant Nicotiana benthamiana and native host sugar beet plants. These vectors can be used to investigate the subcellular co‐localization of multiple proteins in leaf, root and stem tissues of systemically infected plants. Moreover, the BNYVV‐based vectors were used to deliver NbPDS guide RNAs for genome editing in transgenic plants expressing Cas9, which induced a photobleached phenotype in systemically infected leaves. Collectively, the BNYVV‐based vectors will facilitate genomic research and expression of multiple proteins, in sugar beet and related crop plants.     

Production of human growth hormone in transgenic rice seeds: co-introduction of RNA interference cassette for suppressing the gene expression of endogenous storage proteins

Rice seeds are potentially useful hosts for the production of pharmaceutical proteins. However, low yields of recombinant proteins have been observed in many cases because recombinant proteins compete with endogenous storage proteins. Therefore, we attempt to suppress endogenous seed storage proteins by RNA interference (RNAi) to develop rice seeds as a more efficient protein expression system. In this study, human growth hormone (hGH) was expressed in transgenic rice seeds using an endosperm-specific promoter from a 10 kDa rice prolamin gene. In addition, an RNAi cassette for reduction of endogenous storage protein expressions was inserted into the hGH expression construct. Using this system, the expression levels of 13 kDa prolamin and glutelin were effectively suppressed and hGH polypeptides accumulated to 470 μg/g dry weight at the maximum level in transgenic rice seeds. These results suggest that the suppression of endogenous protein gene expression by RNAi could be of great utility for increasing transgene products.     

A high efficient method of constructing recombinant Bombyx mori (silkworm) multiple nucleopolyhedrovirus based on zero‐background Tn7‐mediated transposition in Escherichia coli

A high efficient way for generation of recombinant Bombyx mori (silkworm) multiple nucleopolyhedrovirus by Tn7‐mediated transposition in Escherichia coli was performed. The new system consists of a conditional replication donor vector pRCDM and an attTn7 site blocked E. coli containing BmNPV‐Bacmid. The donor vector contains a replication origin derived from R6Kγ, which propagated only in host cells with pir gene expression decreased in the transposition background greatly. Compared with original vector derived from pUC, the transposition efficiency increased from 5.7 to 66% (≈10 fold) when using conditional replication vector pRCDM transposition into original BmDH10Bac. A further effort to decrease the transposition background was made by blocking the attTn7 site in host E. coli genome. The resulting attTn7 occupied BmDH10BacΔTn7 resulted in a significant increase from 5.7 to 23% (≈4 fold) in the efficacy of generate recombinant BmNPV Bacmid by transposition. Furthermore, the transposition of BmDH10BacΔTn7 with pRCDM resulted typically in 100% white colonies, and it indicated that a zero transposition background was accomplished. This high efficient and zero background transposition system provides a new simple and rapid method for construction of recombinant BmNPV used to express target genes or produce gene‐delivery virus particles in silkworm. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

A DNA replicon system for rapid high‐level production of virus‐like particles in plants

Recombinant virus‐like particles (VLPs) represent a safe and effective vaccine strategy. We previously described a stable transgenic plant system for inexpensive production and oral delivery of VLP vaccines. However, the relatively low‐level antigen accumulation and long‐time frame to produce transgenic plants are the two major roadblocks in the practical development of plant‐based VLP production. In this article, we describe the optimization of geminivirus‐derived DNA replicon vectors for rapid, high‐yield plant‐based production of VLPs. Co‐delivery of bean yellow dwarf virus (BeYDV)‐derived vector and Rep/RepA‐supplying vector by agroinfiltration of Nicotiana benthamiana leaves resulted in efficient replicon amplification and robust protein production within 5 days. Co‐expression of the P19 protein of tomato bush stunt virus, a gene silencing inhibitor, further enhanced VLP accumulation by stabilizing the mRNA. With this system, hepatitis B core antigen (HBc) and Norwalk virus capsid protein (NVCP) were produced at 0.80 and 0.34 mg/g leaf fresh weight, respectively. Sedimentation analysis and electron microscopy of transiently expressed antigens verified the efficient assembly of VLPs. Furthermore, a single replicon vector containing a built‐in Rep/RepA cassette without P19 drove protein expression at similar levels as the three‐component system. These results demonstrate the advantages of fast and high‐level production of VLP‐based vaccines using the BeYDV‐derived DNA replicon system for transient expression in plants. Biotechnol. Bioeng. 2009;103: 706–714. © 2009 Wiley Periodicals, Inc.     

Animal component‐free Agrobacterium tumefaciens cultivation media for better GMP‐compliance increases biomass yield and pharmaceutical protein expression in Nicotiana benthamiana

Transient expression systems allow the rapid production of recombinant proteins in plants. Such systems can be scaled up to several hundred kilograms of biomass, making them suitable for the production of pharmaceutical proteins required at short notice, such as emergency vaccines. However, large‐scale transient expression requires the production of recombinant Agrobacterium tumefaciens strains with the capacity for efficient gene transfer to plant cells. The complex media often used for the cultivation of this species typically include animal‐derived ingredients that can contain human pathogens, thus conflicting with the requirements of good manufacturing practice (GMP). We replaced all the animal‐derived components in yeast extract broth (YEB) cultivation medium with soybean peptone, and then used a design‐of‐experiments approach to optimize the medium composition, increasing the biomass yield while maintaining high levels of transient expression in subsequent infiltration experiments. The resulting plant peptone Agrobacterium medium (PAM) achieved a two‐fold increase in OD₆₀₀ compared to YEB medium during a 4‐L batch fermentation lasting 18 h. Furthermore, the yields of the monoclonal antibody 2G12 and the fluorescent protein DsRed were maintained when the cells were cultivated in PAM rather than YEB. We have thus demonstrated a simple, efficient and scalable method for medium optimization that reduces process time and costs. The final optimized medium for the cultivation of A. tumefaciens completely lacks animal‐derived components, thus facilitating the GMP‐compliant large‐scale transient expression of recombinant proteins in plants.     

O‐Glycosylated 24 kDa human growth hormone has a mucin‐like biantennary disialylated tetrasaccharide attached at Thr‐60

MS was used to characterize the 24 kDa human growth hormone (hGH) glycoprotein isoform and determine the locus of O‐linked oligosaccharide attachment, the oligosaccharide branching topology, and the monosaccharide sequence. MALDI‐TOF/MS and ESI‐MS/MS analyses of glycosylated 24 kDa hGH tryptic peptides showed that this hGH isoform is a product of the hGH normal gene. Analysis of the glycoprotein hydrolysate by high‐performance anion‐exchange chromatography with pulsed amperometric detection and HPLC with fluorescent detection for N‐acetyl neuraminic acid (NeuAc) yielded the oligosaccharide composition (NeuAc₂, N‐acetyl galactosamine₁, Gal₁). After β‐elimination to release the oligosaccharide from glycosylated 24 kDa hGH, collision‐induced dissociation of tryptic glycopeptide T6 indicated that there had been an O‐linked oligosaccharide attached to Thr‐60. The sequence and branching structure of the oligosaccharide were determined by ESI‐MS/MS analysis of tryptic glycopeptide T6. The mucin‐like O‐oligosaccharide sequence linked to Thr‐60 begins with N‐acetyl galactosamine and branches in a bifurcated topology with one appendage consisting of galactose followed by NeuAc and the other consisting of a single NeuAc. The oligosaccharide moiety lies in the high‐affinity binding site 1 structural epitope of hGH that interfaces with both the growth hormone and the prolactin receptors and is predicted to sterically affect receptor interactions and alter the biological actions of hGH.