System For Production Of Recombinant Proteins Research Articles

Scalable protein production by Komagataella phaffii enabled by ARS plasmids and carbon source-based selection

BackgroundMost recombinant Komagataella phaffii (Pichia pastoris) strains for protein production are generated by genomic integration of expression cassettes. The clonal variability in gene copy numbers, integration loci and consequently product titers limit the aptitude for high throughput applications in drug discovery, enzyme engineering or most comparative analyses of genetic elements such as promoters or secretion signals. Circular episomal plasmids with an autonomously replicating sequence (ARS), an alternative which would alleviate some of these limitations, are inherently unstable in K. phaffii. Permanent selection pressure, mostly enabled by antibiotic resistance or auxotrophy markers, is crucial for plasmid maintenance and hardly scalable for production. The establishment and use of extrachromosomal ARS plasmids with key genes of the glycerol metabolism (glycerol kinase 1, GUT1, and triosephosphate isomerase 1, TPI1) as selection markers was investigated to obtain a system with high transformation rates that can be directly used for scalable production processes in lab scale bioreactors.ResultsIn micro-scale deep-well plate experiments, ARS plasmids employing the Ashbya gossypii TEF1 (transcription elongation factor 1) promoter to regulate transcription of the marker gene were found to deliver high transformation efficiencies and the best performances with the reporter protein (CalB, lipase B of Candida antarctica) for both, the GUT1- and TPI1-based, marker systems. The GUT1 marker-bearing strain surpassed the reference strain with integrated expression cassette by 46% upon re-evaluation in shake flask cultures regarding CalB production, while the TPI1 system was slightly less productive compared to the control. In 5 L bioreactor methanol-free fed-batch cultivations, the episomal production system employing the GUT1 marker led to 100% increased CalB activity in the culture supernatant compared to integration construct.ConclusionsFor the first time, a scalable and methanol-independent expression system for recombinant protein production for K. phaffii using episomal expression vectors was demonstrated. Expression of the GUT1 selection marker gene of the new ARS plasmids was refined by employing the TEF1 promoter of A. gossypii. Additionally, the antibiotic-free marker toolbox for K. phaffii was expanded by the TPI1 marker system, which proved to be similarly suited for the use in episomal plasmids as well as integrative expression constructs for the purpose of recombinant protein production.

A Comprehensive Study of MicroRNA in Baculoviruses.

Baculoviruses are viral pathogens that infect different species of Lepidoptera, Diptera, and Hymenoptera, with a global distribution. Due to their biological characteristics and the biotechnological applications derived from these entities, the Baculoviridae family is an important subject of study and manipulation in the natural sciences. With the advent of RNA interference mechanisms, the presence of baculoviral genes that do not code for proteins but instead generate transcripts similar to microRNAs (miRNAs) has been described. These miRNAs are functionally associated with the regulation of gene expression, both in viral and host sequences. This article provides a comprehensive review of miRNA biogenesis, function, and characterization in general, with a specific focus on those identified in baculoviruses. Furthermore, it delves into the specific roles of baculoviral miRNAs in regulating viral and host genes and presents structural and thermodynamic stability studies that are useful for detecting shared characteristics with predictive utility. This review aims to expand our understanding of the baculoviral miRNAome, contributing to improvements in the production of baculovirus-based biopesticides, management of resistance phenomena in pests, enhancement of recombinant protein production systems, and development of diverse and improved BacMam vectors to meet biomedical demands.

Deglycosylated RBD produced in Pichia pastoris as a low-cost sera COVID-19 diagnosis tool and a vaccine candidate.

During the COVID-19 outbreak, numerous tools including protein-based vaccines have been developed. The methylotrophic yeast Pichia pastoris (synonymous to Komagataella phaffii) is an eukaryotic cost-effective and scalable system for recombinant protein production, with the advantages of an efficient secretion system and the protein folding assistance of the secretory pathway of eukaryotic cells. In a previous work, we compared the expression of SARS-CoV-2 Spike Receptor Binding Domain in P. pastoris with that in human cells. Although the size and glycosylation pattern was different between them, their protein structural and conformational features were indistinguishable. Nevertheless, since high mannose glycan extensions in proteins expressed by yeast may be the cause of a nonspecific immune recognition, we deglycosylated RBD in native conditions. This resulted in a highly pure, homogenous, properly folded and monomeric stable protein. This was confirmed by circular dichroism and tryptophan fluorescence spectra and by SEC-HPLC, which were similar to those of RBD proteins produced in yeast or human cells. Deglycosylated RBD was obtained at high yields in a single step, and it was efficient in distinguishing between SARS-CoV-2-negative and positive sera from patients. Moreover, when the deglycosylated variant was used as an immunogen, it elicited a humoral immune response ten times greater than the glycosylated form, producing antibodies with enhanced neutralizing power and eliciting a more robust cellular response. The proposed approach may be used to produce at a low cost, many antigens that require glycosylation to fold and express, but do not require glycans for recognition purposes.

The Influence of Different Culture Media on the Growth and Recombinant Protein Production of Iranian Lizard Leishmania Promastigote.

Leishmania is a eukaryotic protozoan parasite belonging to the Trypanosomatidae family. The Iranian Lizard Leishmania (I.L.L.), which is nonpathogenic to mammals, shows great promise to be used as an expression system for recombinant protein production. Unlike other Leishmania strains, the ideal culture medium for I.L.L. has not been established, although it is commonly cultured in the RPMI1640 medium. We investigated the growth rate of the wild and recombinant I.L.L. in BHI, RPMI1640, LB, and M199 media with and without FBS, hemin, or lyophilized rabbit serum. Subsequently, the expression rate of the recombinant protein in these media was compared. The growth rate of I.L.L. in RPMI1640 medium and LB broth was similar and supplementation with 10% FBS did not affect the growth rate. The amount of protein expression in the LB medium was higher than in the other three media. The LB broth is an appropriate medium for I.L.L. culture and recombinant protein production.

New vectors for urea-inducible recombinant protein production

We have developed a novel urea-inducible recombinant protein production system by exploiting the Proteus mirabilis urease ureR-ureD promoter region and the ureR AraC-family transcriptional regulator. Experiments using the expression of β-galactosidase and green fluorescent protein (GFP) showed that promoter activity is tightly regulated and that varying the concentration of urea can give up to 100-fold induction. Production of proteins of biopharmaceutical interest has been demonstrated, including human growth hormone (hGH), a single chain antibody fragment (scFv) against interleukin-1β and a potential Neisserial vaccine candidate (BamAENm). Expression levels can be fine-tuned by temperature and different urea concentrations, and can be induced with readily available garden fertilisers and even urine. As urea is an inexpensive, stable inducer, a urea-induced expression system has the potential to considerably reduce the costs of large-scale recombinant protein production.

Improvement of recombinant miraculin production in transgenic tomato by crossbreeding-based genetic background modification.

An important optimization step in plant-based recombinant protein production systems is the selection of an appropriate cultivar after a potential host has been determined. Previously, we have shown that transgenic tomatoes of the variety 'Micro-Tom' accumulate incredibly high levels of miraculin (MIR) due to the introduction of MIR gene controlled by a CaMV35S promoter and a heat-shock protein terminator. However, 'Micro-Tom' is unsuitable for commercial production of MIR as it is a dwarf cultivar characterized by small-sized fruit and poor yield. Here, we used the crossbreeding approach to transfer the high MIR accumulation trait of transgenic 'Micro-Tom' tomatoes to 'Natsunokoma' and 'Aichi First', two commercial cultivars producing medium and large fruit sizes, respectively. Fruits of the resultant crossbred lines were larger (~ 95 times), but their miraculin accumulation levels (~ 1,062μg/g fresh mass) were comparable to the donor cultivar, indicating that the high miraculin accumulation trait was preserved regardless of fruit size or cultivar. Further, the transferred trait resulted in a 3-4fold increase in overall miraculin production than that of the previously reported line 5B. These findings demonstrate the effectiveness of crossbreeding in improving MIR production in tomatoes and could pave the way for a more efficient production of recombinant proteins in other plants.

A De Novo Optimized Cell-Free System for the Expression of Soluble and Active Human Tumor Necrosis Factor-Alpha.

Simple SummaryAs a result of increasing demand for the pleiotropic cytokine TNF-α, recombinant human TNF-α protein with appropriate bioactivities was produced in several heterologous in vivo expression systems. While in vivo expression of this cytokine is laborious and lengthy, cell-free or in vitro expression system has the benefits of speed, simplicity, flexibility, focus of all the system energy on target protein synthesis alone, besides high soluble and functional protein yield. Therefore, we employed and optimized an E. coli-based cell-free system for the first time to express recombinant human TNF-α. Our findings revealed that cell-free expression system can be an alternative platform for producing soluble and functionally active recombinant TNF-α with a yield of 390 µg/mL in only 2 h at a temperature of 40 °C for further research and clinical trials.Cell-free (in vitro) expression is a robust alternative platform to the cell-based (in vivo) system for recombinant protein production. Tumor necrosis factor-alpha (TNF-α) is an effective pro-inflammatory cytokine with pleiotropic effects. The aim of the current study was de novo optimized expression of soluble and active human TNF-α by an in vitro method in an E. coli-based cell-free protein synthesis (CFPS) system and its biological activity evaluation. The codon-optimized synthetic human TNF-α gene was constructed by a two-step PCR, cloned into pET101/D-TOPO vector and then expressed by the E. coli CFPS system. Cell-free expression of the soluble protein was optimized using a response surface methodology (RSM). The anticancer activity of purified human TNF-α was assessed against three human cancer cell lines: Caco-2, HepG-2 and MCF-7. Data from RSM revealed that the lowest value (7.2 µg/mL) of cell-free production of recombinant human TNF-α (rhTNF-α) was obtained at a certain incubation time (6 h) and incubation temperature (20 °C), while the highest value (350 µg/mL) was recorded at 4 h and 35 °C. This rhTNF-α showed a significant anticancer potency. Our findings suggest a cell-free expression system as an alternative platform for producing soluble and functionally active recombinant TNF-α for further research and clinical trials.

A Targeted In-Fusion Expression System for Recombinant Protein Production in Bombyx mori.

The domesticated silkworm, Bombyx mori, is an economically important insect that synthesizes large amounts of silk proteins in its silk gland to make cocoons. In recent years, germline transformation strategies advanced the bioengineering of the silk gland as an ideal bioreactor for mass production of recombinant proteins. However, the yield of exogenous proteins varied largely due to the random insertion and gene drift caused by canonical transposon-based transformation, calling for site-specific and stable expression systems. In the current study, we established a targeted in-fusion expression system by using the transcription activator-like effector nuclease (TALEN)-mediated targeted insertion to target genomic locus of sericin, one of the major silk proteins. We successfully generated chimeric Sericin1-EGFP (Ser-2A-EGFP) transformant, producing up to 3.1% (w/w) of EGFP protein in the cocoon shell. With this strategy, we further expressed the medically important human epidermal growth factor (hEGF) and the protein yield in both middle silk glands, and cocoon shells reached to more than 15-fold higher than the canonical piggyBac-based transgenesis. This natural Sericin1 expression system provides a new strategy for producing recombinant proteins by using the silkworm silk gland as the bioreactor.

Protein Expression Optimization Strategies in E. coli: A Tailored Approach in Strain Selection and Parallelizing Expression Conditions.

Escherichia coli remains a traditional and widely used host organism for recombinant protein production. Its well-studied genome, availability of vectors and strains, cheap and relatively straight-forward cultivation methods paired with reported high protein yields are reasons why E. coli is often the first-choice host expression system for recombinant protein production. The chapter enclosed here details protocols and design strategies in strain selection and methods on how to parallelize expression conditions to optimize for soluble target protein expression in E. coli. The methods described have been validated in a protein production research facility.

Genomic diversity, chromosomal rearrangements, and interspecies hybridization in the Ogataea polymorpha species complex.

The methylotrophic yeast Ogataea polymorpha has long been a useful system for recombinant protein production, as well as a model system for methanol metabolism, peroxisome biogenesis, thermotolerance, and nitrate assimilation. It has more recently become an important model for the evolution of mating-type switching. Here, we present a population genomics analysis of 47 isolates within the O. polymorpha species complex, including representatives of the species O. polymorpha, Ogataea parapolymorpha, Ogataea haglerorum, and Ogataea angusta. We found low levels of nucleotide sequence diversity within the O. polymorpha species complex and identified chromosomal rearrangements both within and between species. In addition, we found that one isolate is an interspecies hybrid between O. polymorpha and O. parapolymorpha and present evidence for loss of heterozygosity following hybridization.

Design and expression of a bispecific antibody against dengue and chikungunya virus in plants.

Recombinant proteins have a broad range of applications from basic research to pharmaceutical development. Of utmost importance in the production of recombinant proteins is the selection of the best recombinant protein production system, such that high-quality and functional recombinant proteins are produced. Plants can produce a large quantity of recombinant proteins rapidly and economically. Glycoengineering has created "humanized" plant lines that can produce glycoproteins with specific human glycans with a high level of homogeneity on demand. Here, a detailed protocol was provided to produce a large, multisubunit, and complex bispecific antibody that targets two distinct viruses. The successful production of this multiple-subunit protein demonstrated that plants are the optimal system for the production of recombinant proteins of various sizes and complexity, which can be employed for various applications including diagnostics, therapeutics, and vaccines to combat current and future pandemics.

Structural and functional comparison of SARS-CoV-2-spike receptor binding domain produced in Pichia pastoris and mammalian cells

The yeast Pichia pastoris is a cost-effective and easily scalable system for recombinant protein production. In this work we compared the conformation of the receptor binding domain (RBD) from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Spike protein expressed in P. pastoris and in the well established HEK-293T mammalian cell system. RBD obtained from both yeast and mammalian cells was properly folded, as indicated by UV-absorption, circular dichroism and tryptophan fluorescence. They also had similar stability, as indicated by temperature-induced unfolding (observed Tm were 50 °C and 52 °C for RBD produced in P. pastoris and HEK-293T cells, respectively). Moreover, the stability of both variants was similarly reduced when the ionic strength was increased, in agreement with a computational analysis predicting that a set of ionic interactions may stabilize RBD structure. Further characterization by high-performance liquid chromatography, size-exclusion chromatography and mass spectrometry revealed a higher heterogeneity of RBD expressed in P. pastoris relative to that produced in HEK-293T cells, which disappeared after enzymatic removal of glycans. The production of RBD in P. pastoris was scaled-up in a bioreactor, with yields above 45 mg/L of 90% pure protein, thus potentially allowing large scale immunizations to produce neutralizing antibodies, as well as the large scale production of serological tests for SARS-CoV-2.

Development of a constitutive and an auto-inducible high-yield expression system for recombinant protein production in the microalga Nannochloropsis oceanica

Photoautotrophic microalgae offer a great potential as novel hosts for efficient recombinant protein production. Nannochloropsis oceanica produces an extraordinarily high content of polyunsaturated fatty acids, and its robust growth characteristics, published genome sequence and efficient nuclear transformation make N. oceanica a promising candidate for biotechnological applications. To establish a robust and flexible system for recombinant protein production, we cloned six endogenous, potentially constitutive or inducible promoters from N. oceanica strain CCMP1779 and investigated their strength using monomeric Venus as reporter gene. Microscopic pre-screening of individual transformants revealed that the promoters of elongation factor (EF), tubulin (TUB) and nitrate reductase (NR) enabled high reporter gene expression. Comparative quantitative analyses of transformant populations by flow cytometry and qRT-PCR demonstrated the highest Venus expression from the EF promoter and the NR promoter if extended by an N-terminal 14-amino acid leader sequence. The kinetics of reporter gene expression were analysed during photobioreactor cultivation, achieving Venus yields of 0.3% (for EF) and 4.9% (for NR::LS) of total soluble protein. Since inducible expression systems enable the production of toxic proteins, we developed an auto-induction medium for the NR promoter transformants. By switching the N source from ammonium to nitrate in the presence of low ammonium concentrations, the starting point of Venus induction could be fine-tuned and shifted towards exponential growth phase while maintaining high recombinant protein yields. Taken together, we demonstrate that a model recombinant protein can be produced robustly and at very high levels in N. oceanica not only under constitutive but also under auto-inducible cultivation conditions.Key points• Nannochloropsis oceanica might serve as host for recombinant protein production.• Comparative promoter strength analyses were conducted for twelve different constructs.• Robust high-yield recombinant protein production was achieved under constitutive conditions.• The nitrate reductase promoter enabled protein production under auto-induction conditions.

Plant Molecular Farming: A Viable Platform for Recombinant Biopharmaceutical Production

The demand for recombinant proteins in terms of quality, quantity, and diversity is increasing steadily, which is attracting global attention for the development of new recombinant protein production technologies and the engineering of conventional established expression systems based on bacteria or mammalian cell cultures. Since the advancements of plant genetic engineering in the 1980s, plants have been used for the production of economically valuable, biologically active non-native proteins or biopharmaceuticals, the concept termed as plant molecular farming (PMF). PMF is considered as a cost-effective technology that has grown and advanced tremendously over the past two decades. The development and improvement of the transient expression system has significantly reduced the protein production timeline and greatly improved the protein yield in plants. The major factors that drive the plant-based platform towards potential competitors for the conventional expression system are cost-effectiveness, scalability, flexibility, versatility, and robustness of the system. Many biopharmaceuticals including recombinant vaccine antigens, monoclonal antibodies, and other commercially viable proteins are produced in plants, some of which are in the pre-clinical and clinical pipeline. In this review, we consider the importance of a plant- based production system for recombinant protein production, and its potential to produce biopharmaceuticals is discussed.

Increased brazzein expression by abiotic stress and bioreactor culture system for the production of sweet protein, brazzein

Plant-based recombinant protein production systems are powerful tools that can be used instead of microbial and animal cell culture systems. The aim of this study was to establish a stable brazzein production system from carrot cell suspension using bioreactor. Using Agrobacterium-mediated transformation (stress-inducible SWPA2 promoter and brazzein gene), three transgenic lines (TC1, 11, 12) were generated. During the TC12 culture period, cell proliferation increased rapidly up to 15 days, and the maximum cell division rate (log phase) was reached after 6 days of culture. Fresh weight peaked at day 18, 3.8-fold (7.6 g 100 mL−1) higher than on day 1, and gene expression was tenfold higher on day 27 than on day 0. The brazzein-encoding gene was also analyzed after treatment with various stress factors for increase of brazzein gene expression, and abscisic acid (ABA) and hydrogen peroxide (H2O2) proved most effective. In particular, the gene expression was enhanced 2.5-fold with 220 μM H2O2 and 2.8-fold with 50 μM ABA, compared with controls. Transgenic cells were then transferred to various types of air-lift bioreactor, and the column bioreactor achieved a higher biomass (238.9 g L−1) than cone and balloon bioreactors. These results demonstrate an efficient protein production system for brazzein using an air-lift bioreactor that may be suitable for the use in the food industry.

An overview of yeast cell wall proteins and their contribution in yeast display system

Yeast surface display has become an increasingly popular tool for protein engineering and library screening applications. Although, recent advances have greatly expanded the capability of yeast surface display, the protein display system is still far away from industrial application. One of the major components of a stable, efficient and successful yeast surface display system is cell wall anchor protein with which our desired foreign protein will be attached. We studied 80 different yeast cell wall anchored proteins originated mostly from Saccharomyces cerevisiae and Candida albicans. We studied in details all the cell wall proteins in order to find out suitable cell wall proteins to recommend for the researchers to use in the construction of yeast display system. We considered selective physical properties of different yeast cell wall proteins that are crucial for selecting best suited cell surface anchor proteins which are molecular weight, binding domain of anchor protein, length of amino acid and fusion site. Finally, our studies showed that Ccw11, Ccw12. Cwp1, Cwp2, Dan1, Gas1, Gas5, Exg1, Ycr89, Ecm33, Pga4, Sap9, Sap10, Pst1, Pir1, Pir2, Pir3, Pir4, Cis1, Scw4, Scw6, Bgl2, Uth1, Scw1 are the promising and suitable cell wall anchor proteins could be used in construction of yeast cell surface display system. Additionally, this review presents detailed information about all the cell wall proteins in a single work. The future researchers in this field will be able to construct more efficient yeast display system for recombinant protein production at industrial scale using the knowledge presented in this work.
 Asian J. Med. Biol. Res. June 2019, 5(4): 246-257

Establishment of DHFR-deficient HEK293 cells for high yield of therapeutic glycoproteins

Since the use of protein therapeutics is effective for treating intractable human diseases, the production of biologic therapeutic agents has dramatically increased over the past three decades. The Chinese hamster ovary (CHO) cell lines are the most commonly used host cell expression system for recombinant protein production. High productive and stable clonal cell lines for recombinant protein production have been established from the DHFR-deficient CHO cell using the dihydrofolate reductase/methotrexate (DHFR/MTX) selection methods. Human embryonic kidney 293 (HEK293) cells are alternative host cells widely used for protein production. In most case, however, the cells are used for the transient expression, and there is no gene amplification system in HEK293 cells. In this study, we established a DHFR-deficient HEK293 cell line for the high yield of recombinant proteins. We doubly knocked out DHFR and DHFR2 in the MAN1A1/A2/B1/C1-quadruple knockout HEK293 (QD-KO) cells, using the CRISPR/Cas9 system. The DHFR-deficient QD-KO cells were used to overexpress two proteins, lysosomal acid lipase and the constant fragment of human immunoglobulin G1 by the DHFR/MTX gene-amplification method. This method resulted in a dramatic increase in the two protein expressions in the DHFR-deficient QD-KO cells by increasing MTX concentration. Our system could be adopted in the production of several recombinant proteins including therapeutic proteins.

Pichia pastoris Promoters.

The methylotrophic yeast Pichia pastoris (Komagataella phaffii) is used as an expression system for recombinant protein production for a variety of applications. It grows rapidly on inexpensive media containing methanol, glucose, glycerol, or ethanol as a sole carbon source. P. pastoris makes many posttranslational modifications and produces recombinant proteins either intracellularly or extracellularly. Because of these properties, P. pastoris has become a highly preferred host organism for biotechnology, pharmaceutical industry, and researchers.Recombinant protein production is usually performed under the control of the promoter of the alcohol oxidase gene I (AOX1). The AOX1 promoter is induced by methanol and repressed by glucose and ethanol. The regulation mechanisms of the AOX1 promoter have been studied in recent years. Another promoter used in recombinant protein production is derived from glyceraldehyde 3-phosphate dehydrogenase (GAP). It is a constitutive promoter. Recent literature showed that newly identified promoters of P. pastoris are promising as well, in addition to pAOX1 and pGAP.In this chapter, the regulation mechanisms of inducible pAOX1 and constitutive pGAP promoters are discussed. In addition, here we present an overview about the novel ADH3 promoter and alternative promoters of P. pastoris.

Efficient Development of a Mixed Feed Process for Pichia pastoris.

Pichia pastoris is one of the most important host organisms for the recombinant production of proteins in industrial biotechnology. A prominent promoter system for recombinant protein production in P. pastoris is the promoter of alcohol oxidase (PAOX1) which is induced by methanol, but repressed by several other carbon sources, like glucose and glycerol. Thus, typical cultivation strategies for such P. pastoris strains describe two different phases: growth on a carbon source, like glycerol, to get a high biomass concentration, followed by the induction of recombinant protein production by methanol. However, cells barely grow on methanol resulting in only moderate productivity in such bioprocesses. To enhance productivity, it is common to employ mixed substrate feeding strategies. The knowledge of certain strain-specific parameters is required to be able to set up such mixed feed fed-batch cultivations to avoid methanol accumulation and guarantee highest productivity. Here, we present an efficient strategy comprising only one experiment to determine the settings of such a mixed feed system based on the physiology of the respective yeast strain.

Construction of a Platform for the Development of Pharmaceutical and Medical Applications Using Transgenic Silkworms

We have been constructing a platform for the development of pharmaceutical and medical applications using the domesticated silkworm, Bombyx mori, as a new animal model for drug development and evaluation. Because silkworm larvae originally have the capacity to synthesize up to 0.5 g of silk proteins, genetically modified silkworms (transgenic silkworms) are expected to have high potential in the production of recombinant silks/proteins. An innovative method for generating transgenic silkworms was established in 2000, and ever since this epoch-defining technological development, longstanding efforts have succeeded in developing novel silks that enable the manufacture of new textile materials for regenerative medical uses. Furthermore, we have succeeded in developing a new system of recombinant protein production. This recombinant protein production system is currently capable of producing a maximum of approximately 15 mg recombinant protein per silkworm larva. Transgenic silkworms have also been shown to produce a wide variety of useful proteins, including antibodies and membrane proteins. Some of these recombinant proteins have been in commercial use since 2011. In addition, we have been developing transgenic silkworms as a novel animal model for testing medicines based on metabolic similarities between silkworms and mammals. These applications show the suitability and potential of transgenic silkworms for medical use. Here, we will describe the challenges faced in creating a transgenic silkworm-based platform for pharmaceutical and medical applications.