E. coli vs. Yeast in Protein Expression

Research will be limited to:
- Organisms: E. coli
- Organisms: Yeast
- Focus: Protein Expression
- Topics: Post-translational Modifications
- Topics: Yield
- Topics: Cost
- Topics: Timeline

Strategy: I will conduct a comparative analysis focusing on the four key metrics (PTMs, yield, cost, and timeline) for E. coli versus yeast (specifically S. cerevisiae and P. pastoris) expression systems. The search will target recent review articles and industrial case studies that provide quantitative benchmarks for production timelines and cost-of-goods (COGS) comparisons.
Queries:
- comparison E. coli vs yeast protein expression yield cost timeline
- post-translational modifications E. coli vs yeast recombinant proteins
- economic analysis microbial protein production E. coli yeast cost per gram
- recombinant protein production timeline E. coli vs Pichia pastoris vs Saccharomyces cerevisiae
- glycosylation patterns yeast vs E. coli protein expression systems

Executing 5 concurrent searches:
- comparison E. coli vs yeast protein expression yield cost timeline
- post-translational modifications E. coli vs yeast recombinant proteins
- economic analysis microbial protein production E. coli yeast cost per gram
- recombinant protein production timeline E. coli vs Pichia pastoris vs Saccharomyces cerevisiae
- glycosylation patterns yeast vs E. coli protein expression systems

Found 5 papers. Key excerpts:

Host expression system modulates recombinant Hsp70 activity through post-translational modifications — Mauricio M. Rigo et al., 2020

The use of model organisms for recombinant protein production results in the addition of model-specific post-translational modifications (PTMs) that can affect the structure, charge, and function of the protein. The 70-kDa heat shock proteins (Hsp70) were originally described as intracellular chaperones, with ATPase and foldase activity. More recently, new extracellular activities of Hsp70 proteins (e.g., as immunomodulators) have been identified. While some studies indicate an inflammatory potential for extracellular Hsp70 proteins, others suggest an immunosuppressive activity. We hypothesized that the production of recombinant Hsp70 in different expression systems would result in the addition of different PTMs, perhaps explaining at least some of these opposing immunological outcomes. We produced and purified Mycobacterium tuberculosis DnaK from two different systems, Escherichia coli and Pichia pastoris, and analyzed by mass spectrometry of the protein preparations, investigating the impact of PTMs in an in silico and in vitro perspective. The comparisons of DnaK structures in silico highlighted that electrostatic and topographical differences exist that are dependent upon the expression system. Production of DnaK in the eukaryotic system dramatically affected its ATPase activity and significantly altered its ability to downregulate MHC II and CD86 expression on murine dendritic cells (DCs). Phosphatase treatment of DnaK indicated that some of these differences related specifically to phosphorylation. Altogether, our data indicate that PTMs are an important characteristic of the expression system, with differences that impact interactions of Hsps with their ligands and subsequent functional activities.
Database

           Mass spectrometry proteomic data are available in the PRIDE database under the accession number PXD011583.

Introduction

     Heat shock protein 70s (Hsp70s) are a highly conserved class of molecular chaperones that fold a large proportion of the proteome [[1-4]]. Hsp70 is comprised of four major structural domains: a region that binds ATP called nucleotide binding domain (NBD); a

region that binds client proteins (usually hydrophobic peptides) referred to as substrate binding domain (SBD); a linker segment of few residues that physically connects NBD and SBD; and a C-terminal region that functions as a ‘lid’, participating in Hsp70 substrate turnover. The Hsp70 mode of action favors the existence of two extreme conformations: an open state, in which the ATP is bound and the affinity for the substrate is low; and a closed state, in which ATP hydrolysis favors substrate binding to the SBD [[5, 6]].

     Hsp70 has been extensively studied; however, little is known about the post-translation modifications (PTMs) of Hsp70 and how they might affect its function. Global proteomic studies have uncovered a wide range of PTMs on Hsp70 isoforms in different organisms that include phosphorylation, acetylation, and ubiquitylation. Recent evidence suggests that even single PTMs on Hsp70 can dramatically alter ATPase activity, folding of clients, and localization in the cell [[7-9]]. It is becoming clear that these modifications form a ‘chaperone code’ that acts as a crossroads for cellular signaling and fine-tunes Hsp70 function [[10, 11]].

     Although heat shock proteins (Hsps) were described originally as intracellular proteins (e.g., cytosol, nucleus, endoplasmic reticulum) [[12-14]], they were later found to be also present in the extracellular milieu [[15, 16]], secreted by a nonclassical route [[17, 18]]. The extracellular roles of Hsps are not completely understood. For example, studies investigating the inflammatory properties of extracellular Hsps have on occasion reported opposing activities of Hsp70 proteins that have led to some ambiguity in the field. While some describe an immunostimulatory role for extracellular mammalian Hsp70 [[19]], results by other groups demonstrate this observation is at least in part explainable by bacterial contaminants [[20, 21]]. However, while most studies of the potential immunomodulatory roles of Hsp70 carried out in

vivo indicate a pro-inflammatory effect [[22]], studies utilizing DnaK (bacterial Hsp70) purified from Mycobacterium tuberculosis (M. tuberculosis) demonstrated an anti-inflammatory effect [[23]].

     Studies on extracellular functions of Hsp70s commonly utilize proteins obtained from different sources. Model organisms used for recombinant protein production such as Escherichia coli and Pichia pastoris (P. pastoris) have unique advantages that include ease of use, cellular doubling time, cost, and scalability [[24]]. Importantly, although some E. coli proteins were already identified with the presence of serine, threonine, or tyrosine phosphorylation [[25, 26]], this system generally does not carry out these PTMs, a benefit or disadvantage depending on the desired downstream application of the recombinant protein. It is possible that some of the contradictory results observed in the literature using recombinant Hsp70 could at least in part be explained by host-induced PTMs.

     In this study, we hypothesized that the production of recombinant Hsp70 in different expression systems would result in the addition of different PTMs. To characterize differences in Hsp70 PTMs created by expression in different hosts, we used mass spectrometry to analyze protein preparations of DnaK from M. tuberculosis expressed in two different expression systems (E. coli and P. pastoris). DnaK from M. tuberculosis is largely used as an Hsp70 model due to structural homology with Hsp70 proteins from other organisms; also, the sequence similarity after a pairwise alignment with DnaK from E. coli is over 70% (UniProt ID P9WMJ9 and P0A6Y8 submitted to EMBOSS-NEEDLE server). We analyzed the impact of PTMs in an in silico perspective in a complete DnaK structure obtained from homology modeling. Functional analysis of each DnaK preparation was evaluated in ATPase activity assays, and also in immunomodulation assays, assessing their ability to reduce MHC II and CD86 expression in dendritic cells [[27]].

Targeted Deletion of Los1 Homologue Affects the Production of a Recombinant Model Protein in Pichia pastoris. — Najmeh Zarei et al., 2021

The methylotrophic yeast Pichia pastoris is frequently used for the high level production of recombinant proteins, especially where the Escherichia coli system fails to deliver correctly folded functional proteins, or the Saccharomyces cerevisiae system produces hyper-mannosylated inactive proteins [ 1 ] . The recent development of glycoengineered Pichia strains generating human-like glycan pattern, along with the adaptation of new genome editing tools, such as CRISPR system, has offered further advantages for the industrial application of Pichia platform [ 2 , 3 ] .
Available reports indicate the highest amount of intracellular (22 g/L) and extracellular (18 g/L) protein expression levels in P. pastoris [ 4 ] . As a matter of fact, not all proteins are produced at such a high level, and the amount of protein expression depends largely on the intrinsic properties of the target protein. Fortunately, in many cases, the initial expression levels can greatly be improved by the manipulation of host genome or culture conditions. The genetic manipulation of host cells using targeted gene deletion or overexpression has been one of the most important optimization approaches that can affect the quantity and quality of a desired protein product [ 5 ] .
The production of recombinant proteins in P. pastoris is a growth-dependent process [ 6 ] such that host cell engineering for cell lifespan extension is considered as a promising strategy in increasing the yield of recombinant protein production. Cell aging can affect both protein synthesis and degradation, by altering the components of the translation machinery, as well as the intracellular distribution of newly synthesized proteins [ 7 , 8 ] . In this sense, the manipulation of aging process may alter the final yield of recombinant products during the mass production procedures.
The budding yeast, S. cerevisiae , has been an excellent model for aging and lifespan-related studies. Both RLS (the number of daughter cells generated by a mother cell before replicative senescence) and CLS (the survival time of a yeast cell in non-dividing condition) have already been defined, and molecular mechanisms involved in the yeast lifespan have been investigated in details [ 9 , 10 ] . So far, several genes identified in yeast have been shown to be able to modulate the aging process through different mechanisms [ 11 , 12 ] . Los1 (loss of supression), as a non-essential gene in S. cerevisiae ,

encodes a nuclear tRNA exporter, and its deletion robustly increases lifespan in this organism [ 12 ] . As a member of the conserved β-importin family, Los1 dictates the direction of tRNA transport across nuclear pores using the nuclear-cytoplasmic gradient of RanGTP [ 13 ] . Considering the conserved mechanisms influencing the lifespan and aging among eukaryotes [ 12 , 14 , 15 ] , herein, we report the inactivation of Los1 gene homologue, in P. pastoris using a site-directed disruption construct. The impact of this deletion on the viability and growth of the deletant was monitored, and the production yield of a model recombinant protein, anti-CD22 scFv, was investigated.
Strains, plasmids, and culture conditions
E. coli Top 10F′ was employed as a host for DNA manipulations, and P. pastoris strain GS115 (his4) with mut + phenotype (Invitrogen, San Diego, CA, USA) was used for protein expression studies. pGEM-T Easy Vector (Promega, Madison, USA) was applied for cloning of PCR products, and the pSH67 plasmid, carrying G418 resistance gene (KanMX), as a yeast selection marker, was utilized as an intermediate vector for assembling the los1 disruption cassette ( Fig. 1 ). E. coli Top 10F′ was cultured in Luria-Bertani medium (in w/v; 1% tryptone, 0.5% yeast extract, and 1% NaCl, pH 7.0) at 37 °C. Ampicillin was added to the LB medium for plasmid selection at the final concentration of 100 μg/ml. The yeast P. pastoris strain was grown in YPD medium (in w/v; 1% yeast extract, 2% peptone, and 2% dextrose), whereas YPD plates containing G418 (300 μg/mL or as indicated) were used for the selection of G418-positive P. pastoris transformants. The BMGY was prepared with 2% peptone, 1% yeast extract, 1% glycerol, 1.34% yeast nitrogen base, with ammonium sulfate but without amino acids, and 4 × 10 -5 % biotin in 100 mM potassium phosphate buffer. The preparation of BMMY was carried out the same as B

MGY, except for the replacement of glycerol with 0.5 % methanol.
Identification of Los1 homologue in P. pastoris
For the identification of the Los1 homologue in P. pastoris , the amino acid sequence of S. cerevisiae Los1 was retrieved from the UniProt database, and a protein blast against fungal database was performed (https://www.uniprot.org/). Similar searches were also carried out at http://blast.ncbi.nlm.nih.gov and http://pichiagenome-ext.boku.ac.at/. Phylogenomic databases such as eggNOG (http://eggnogdb.embl.de), HOGENOM (http://hogenom.univ-lyon1.fr/), OrthoDB (https://www.orthodb.org/) and Inparanoid (http:// inparanoid.sbc.su.se/cgi-bin/index.cgi) were also used to investigate the presence of the Los1 homologue in P. pastoris . Nuclear localization signal sequence was predicted using an online software (http://mleg.cse.sc. edu/seqNLS/) .
Preparation of Los1 gene disruption cassette
The target gene disruption construct was prepared using standard PCR and cloning protocols, and the final cassette was validated by restriction mapping and DNA sequencing [ 16 ] . Briefly, to facilitate homologous recombination, a 1000-bp upstream and a 1000-bp downstream flanking regions of Los1 homologue in P. pastoris were amplified using specific primer sets KO-UP_F/R and KO_DW_F/R, harboring Sac I/ Pvu II and Xba I/ Bgl II restriction sites, respectively ( Table 1 ). The amplified fragments were sequentially subcloned into pSH67, the upstream and downstream of KanMX selection marker, to create the pSH67- los1 -KO construct ( Fig. 1A and 1B ).
Transformation of P. pastoris and selection of disruptant clones
The pSH67- Los1 -KO construct was double-digested with Xho I /Pvu II enzymes, and the linearized Los1 -KO fragment, containing KanMX and flanking los1 up and down sequences,

Pichia pastoris versus Saccharomyces cerevisiae: a case study on the recombinant production of human granulocyte-macrophage colony-stimulating factor — Anh-Minh Tran et al., 2017

Background Human granulocyte-macrophage colony-stimulating factor (hGM-CSF), a glycosylated cytokine, plays a vital role in proliferation and differentiation of granulocytes and macrophages from bone marrow progenitor cells [ 1 ]. hGM-CSF also enhances cytotoxicity of macrophages and monocytes towards tumor cells [ 2 ], as well as tumor presentation of dendritic cells [ 1 , 3 ]. Due to its stimulatory capacity on hematopoietic stem cells, recombinant hGM-CSF (rhGM-CSF) is recommended for therapeutic use in combination with chemo- or radio-therapy for cancer or transplantation patients [ 4 ]. hGM-CSF comprises 127 amino acids. The presence of 2 N - and 4 O -glycosylation sites leads to different glycoforms with molecular weights ranging from 14 to 60 kDa [ 3 ]. The glycosylation of the protein affects its pharmacokinetics, in vivo half-life, immunogenicity and cytotoxicity [ 3 ]. rhGM-CSF is produced from bacteria, baker yeast or mammalian hosts [ 5 ]. rhGM-CSF produced in Escherichia coli is biologically active but unstable in human plasma. It induces an immune reaction because N-formyl methionine (fMet) is its first amino acid. rhGM-CSF produced in mammalian hosts has a similar glycosylation pattern to the native human protein, but production rates are slow [ 6 , 7 ]. The product from baker’s yeast, Saccharomyces cerevisiae , is glycosylated and approved by the FDA for the treatment of neutropenia and leukemia in combination with chemo- or radio-therapy for cancer or transplantation patients. The drawbacks of using S. cerevisiae are hyper-glycosylated products and low cell density growth. The methylotrophic yeast Pichia pastoris has emerged as an alternative host due to its shorter and less immunogenic glycans, higher density cell growth and higher secreted protein yields than S. cerevisiae . In this study, we show that P. pastoris secretes a higher yield of more active recombinant rhGM-CSF than S. cerevisiae .
Methods In silico codon optimization hGM-CSF coding sequences were from Gene Bank (Accession M11220).

In silico codon optimization was done using DNA 2.0 software with P. pastoris and S. cerevisiae codon tables and optimized parameters [ 8 ]. The optimization evaluation was done via http://www.gcua.schoedl.de with low (<20%) frequency codon usage displayed as hatched bars and very low (<10%) frequency codon usage as white bars. Expression of rhGM-CSF in P. pastoris and S. cerevisiae Recombinant P. pastoris and S. cerevisiae rhGM-CSF clones were expressed as described previously [ 9 , 10 ]. The expression of the rhGM-CSF was analyzed by SDS-PAGE and immuno blot probed with an antibody against hGM-CSF (LifeSpan BioScience). Briefly, recombinant S. cerevisiae strain BY4742/ hgm - csf was cultured in selective CSM-ura medium (6.7 g/L yeast nitrogen base with ammonium sulphate, 0.1 M sodium phosphate, supplemented with amino acids lacking uracil [ 11 ]) supplemented with 2% glucose until the OD 600 of the culture was 0.5–1. The cells were then harvested and transferred into CSMG-ura medium (CSM supplemented with 20 g/L galactose) with shaking at 230 rpm at 30 °C. The pH 6.0 of each medium was stabilized by 0.1 M sodium phosphate buffer. The sample supernatants were collected after 72 h of induction and stored for further analysis. Recombinant P. pastoris X33:: hgm - csf was grown in 10 mL BMGY medium (2% peptone, 1% yeast extract, 0.34% yeast nitrogen base, 1% ammonium sulfate, 1% glycerol, and 0.4 mg/L biotin, buffered with 1/10 volume of pH 6.0 potassium phosphate buffer) at 30°C with constant shaking at 250 rpm until the culture reached an OD 600 of 2–4. The cells were harvested and re-suspended in 10 mL BMMY (0.5% methanol is substituted for 1% glycerol in BMGY) with the same growth conditions. For induction, methanol was added every 24 h to a final concentration of 0.5%.

After 72 h, culture supernatants were collected by centrifugation for further analysis. Fermentation of recombinant P. pastoris and S. cerevisiae Recombinant yeast S. cerevisiae strain BY4742/ hgm - csf was batch-cultured in selective CSM(-ura) medium, pH 5.0 supplemented with 2% glucose until the culture reached an OD 600 of 0.5 to 1, and then transferred into 3.18 L YP medium to a final OD 600 of 0.5 in a 5 L LiFlus-GX fermenting vessel system (Biotron). Others growth parameters were temperature at 30 °C, aeration rate 1.5 vvm (through a filter), and agitation speed 720 rpm. Every 24 h, 40 g/L galactose was added at a rate of 6.7 mL/h. Samples were harvested at every 4 h and analyzed. Growth of the yeast cultures was monitored by optical density measurements at 600 nm. Protein secretion in the fermented medium was analyzed by SDS-PAGE and the protein concentration was determined using Bradford’s method [ 12 ]. The supernatant was stored at −20 °C for later purification. For P. pastoris expression [ 13 ], a flask containing 150 mL BMGY containing 0.4 µg/mL biotin was inoculated from a frozen glycerol stock. The inoculum seed flask was grown at 30 °C, 250 rpm, and 24 h until OD 600 = 2–6. A sterilized fermenter containing 2.5 L Fermentation Basal Salts medium supplemented with 4% glycerol and 11 mL PTM 1 trace salts was prepared, the pH adjusted to 5.0 with ammonium hydroxide, the temperature set to 30 °C, agitation at 750 rpm and aeration to 5.0 vvm air. The culture in the inoculum seed flask was completely transferred into the fermenter in which the batch culture was grown for 24 h, until the glycerol was completely consumed. Glycerol feeding was initiated for about 4 h at a feed rate to 18.15 mL/h 50% w/v glycerol containing 1.2% v/v PTM 1 trace salts. Methanol feeding was then initiated for 72

Non-Mammalian Eukaryotic Expression Systems Yeast and Fungi in the Production of Biologics. — Mary Garvey, 2022

The manufacture of biologics is a rapidly growing industry as these specific therapeutics offer targeted treatment approach for many chronic and prevalent medical conditions including cancer, cardiac disease, neurological disease and autoimmunity. According to the Food and Drug Administration (FDA), a biologic is a therapeutic substance produced by a biological process using biological systems as opposed to the process of chemical synthesis (small molecules) and includes vaccines, antibody therapies, non-vaccine therapeutic immunotherapies, gene therapies and cell therapy [ 1 ]. While both production systems have definitive advantages ( Table 1 ), biologics differ from synthetic small molecule drugs in terms of cost, production, administration, and clinical efficacy. The use of biotechnology and recombinant technology to manufacture therapeutic biologics relies on the use of living systems, molecular engineering and bioreactors (typically submerged state fermentations) to produce large molecules displaying desired biological activity. Living systems in use as biologic production platforms at industrial scale include prokaryotic bacterial species, e.g., Escherichia coli , eukaryotic yeast and fungal systems, e.g., Saccharomyces cerevisiae , Aspergillus , plant systems, insect systems, mammalian and human expression systems and cell lines [ 2 ]. Currently, small molecules account for 90% of global therapeutic sales as they are used for the treatment of chronic conditions, the biologics market however, is increasing [ 3 ]. The biologics market is predicted to reach $580.5 billion (EUR 513.5 billion) by 2026, from a cumulative sales value of $652 billion from 2014 to 2017 [ 4 ]. Indeed, Recombinant DNA (RDNA) technology has enabled the production of many biologically active proteins used in disease prevention, treatment and management [ 4 ]. The biologic Humira (by AbbVie), a recombinant monoclonal antibody (Mab) used to treat autoimmune disease is currently the highest selling therapeutic globally, generating 60% of AbbVie’s revenue [ 5 ]. In cancer therapy, biologics such as Herceptin offer treatment options currently unmet as potent anticancer agents in therapeutic cocktails [ 6 ]. More recently, Chimeric antigen receptor (CAR) T cell therapy has emerged as a game changer in cancer treatment. CAR T cell therapy is based on genetically engineering patient T cells to selectively attack cancer cells expressing a specific target antigen [ 7 ]. Recently, advances

are also being made in the application of Cell-free systems for the production of biologics without using living cells [ 4 ].
Bioprocessing occurs in 4 phases: strain/cell line selection and propagation, upstream processing (fermentation), downstream processing, and drug formulation with one biologic usually produced from 1 cell strain [ 8 ]. The bioprocessing systems in use for the production of many biologics typically use mammalian Chinese hamster ovary (CHO) cells and murine myeloma cells, with a recent shift towards the use of human derived cell lines [ 2 ] due to the ease of post translational modifications (PTMs). PTMs play a vital role in biological processes functioning in many molecular pathways, where PTM errors are observed in many disease states [ 9 ]. Fungal and yeast cell systems however, offer many advantages as expressions systems for numerous biologic types. Yeast expressions systems are robust, amenable to genetic engineering or genetic modification (GM), cost-effective, possess native PTM machinery, and do not release endotoxins during processing [ 10 ]. Indeed, yeast demonstrate prokaryotic (rapid cell division, single cells, ease of growth) and eukaryotic features (cell organelle, PTM activity) simultaneously making them ideal candidates in the manufacture of recombinant proteins [ 11 ]. Features including low production cost, high titre value, pyrogen free, and current classification as Generally Recognised As Safe (GRAS) organisms [ 10 ]. Fungal strains of species including Aspergillus and Penicillium are considered as GRAS by the FDA and are used as expression systems by many biotechnology companies to produce varied biological products [ 12 ]. In contrast, to the unicellular yeast systems however, filamentous fungi have complex morphological features in submerged cultures which can be challenging for industrial scale up [ 13 ]. This review outlines the application of yeast and fungal cells as platforms for the production of biologics.
Eukaryotic cell lines, including CHO cells, human cells and insect cells, are invaluable expression systems for the production of many recombinant proteins [ 14 ]. Mammalian cell lines of animal and human origin however, are costly and prone to microbial contamination issues with viral species representing the greatest treat [ 15 ]. With advances in recombinant protein technology, expression of recombinant protein-based biopharmaceuticals in prokaryotic and non-mammalian euk

aryotic cells has become cheaper, more productive, promoting the industrial production of many biologics at industrial scale. Unlike eukaryotic systems, prokaryotic expression systems often produce proteins which do not fold properly, are inactive, produce endotoxins and proteins not amenable to PTM [ 16 ]. PTM involves any process which alters the protein composition and includes the irreversible or reversible addition of a chemical group, e.g., phosphate, carbohydrates termed glycosylation, and polypeptides in ubiquitylation [ 9 ]. Such alterations are often related to the biological activity of the protein due to improper folding and its direction within the cells, where a loss of functionality may occur [ 17 ]. Glycosylation in particular is of significance, as ca. 60% of protein biologics are therapeutic glycoproteins [ 18 ]. It is also noteworthy that the over glycosylation of proteins can negatively impact enzyme activity, including enzyme binding and protein stability [ 19 ]. PTMs takes place in several cell organelles including the nucleus, cytoplasm, endoplasmic reticulum (ER) and Golgi apparatus [ 9 ]. Proteins produced via prokaryotic expressions systems therefore, must pass through an in vitro process for the insertion of PTM adding steps during the synthesis, increasing costs and reducing yield [ 18 ]. Additionally, yeasts have a high robustness and tolerance of the harsh fermentation conditions present in bioreactors and bioprocessing scale up [ 20 ]. Non-mammalian eukaryotic systems (yeast and fungi) therefore, have clear advantages over prokaryotic systems ( Table 2 ).
Yeast are single celled microorganisms within the Fungus kingdom, being defined as unicellular fungi. Yeast are eukaryotic microbial species having a cell wall and membrane bound organelle unlike bacteria being prokaryotic. Fungi growing as a yeast morphology have historically been used in the production of food and beverages [ 25 ]. Due to their eukaryotic nature, yeast have also long been established as models for the study of mammalian cells, biochemical pathways and evolution. As host expression systems yeast have the advantages of rapid growth, high cell density, relatively inexpensive media requirements, and ease of genetic manipulation found in bacteria coupled with the ability of post-translational modifications, such as proteolytic processing, folding, disulfide bond formation and glycosyl

Bacterial expression systems for recombinant protein production: E. coli and beyond. — Rachel Chen, 2012

Introduction

Recombinant protein production is an enormous field. There seems no sign that the expansion of this field will abate anytime soon. Impressive progresses in the recombinant protein technology over the past decades have brought hundreds of therapeutic proteins into clinical applications. As there are hundreds more therapeutic proteins in clinical trials, researches aimed to better the technology will continue to speed ahead. The advent of systems biology era is an important driving force that propels the field. The desire to understand the functions of hundreds and thousands of proteins, whose sequences were just recently made available, demands new approaches that deliver each and every single protein (including membrane proteins) in quantities and quality dictated by the structural and biochemical analysis. Moreover, the inherent biodiversity in both peptide sequences and post-translational modifications adds significantly to the complexity of the task to deliver a bioactive protein, because it requires not only the synthesis of peptide backbone but also its authentic modification. Many ingenious strategies were developed over the recent years to meet these challenges. This review hopes to capture some of the newest development.
Understandably, providing a review for such a broad field is difficult, even with the confine of prokaryotic expression systems. To keep the task manageable, this review will cover references from 2006 with only a few exceptions, where continuity demands inclusion of these older references. Many excellent reviews have been published over the recent years (Demain and Vaishnav, 2009;Makino et al., 2011a;Zerbs et al., 2009). In order to make this review a meaningful contribution to the field, I intend to complement the existing reviews by focusing on areas that have received less coverage in other recent reviews. Three bacterial expression systems are reviewed in some details, Escherichia coli, Lactoccocus lactis, and Pseudomonas. Bacillus system is not reviewed here as excellent reviews are published recently (Nijland and Kuipers, 2008;Pohl and Harwood, 2010). Only selected aspects are reviewed for each chosen system. For example, in E. coli the focus is on posttranslational modification and trend of developing antibiotic-free selection systems. On L. lactis, membrane protein expression is emphasized. Where possible, a comparison to E. coli system is made. Other important developments are only briefly mentioned in the review and recent reviews are referenced.
2. E. coli systems: new development E. coli expression system continues to dominate the bacterial

expression systems and remain to be the first choice for laboratory investigations and initial development in commercial activities or as a useful benchmark for comparison among various expression platforms. E. coli system is also the basis for efforts in protein engineering and high-throughput structural analysis ( Gordon et al., 2008;Koehn and Hunt, 2009). Reviews also appeared recently on general aspects of using the E. coli system and more specific topics such as single-domain antibody fragments (De Marco, 2011;Koehn and Biotechnology Advances 30 (2012) Hunt, 2009), stress and stress responses associated with recombinant protein production ( Sevastsyanovich et al., 2010).
Extracellular production of proteins is highly desirable as it could greatly reduce the complexity of a bioprocess and improve product quality. The inability of E. coli in secretion of protein products to growth medium has long been considered as a major drawback of the system. However, the dogma that E. coli secretes no protein has been challenged recently by the recognition of both its natural ability to secrete protein in common laboratory strains and increased ability to secrete proteins in engineered cells. Significant efforts have been made in recent years to address this issue (Ni and Chen, 2009). A survey of recent literature shows that the strategies fall into four categories, 1) engineering dedicated secretion systems that naturally exist in pathogen E. coli; 2) use carrier proteins with no known translocation mechanisms; 3) use cell envelope mutants; and 4) co-expression a lysis-promoting protein (Kil) (Ni and Chen, 2009). Efforts in enhancing its ability to secrete proteins to growth medium are significant and some successful technology has begun to impact the production beyond laboratory investigations. Additional reviews on the topic include the review on using autotransporters ( Jong et al., 2010).
While potential problems of using antibiotic and antibiotic-resistance markers in large scale recombinant protein production have long been recognized, only recently, efforts in developing alternative methods for selections have born fruits in practical sense. Additionally, engineering glycosylation and other post-translational modification into the system is an exciting development in recent years. These two areas will be reviewed in the following sections.

Glycosylation and other post-translational modifications

Just a few years ago, it was widely believed that bacteria were incapable of making glycosylated proteins. The discovery of N-linked

glycosylation system in a Gram-negative bacterium Campylobacter jejuni and subsequent transfer of the system to E. coli ( Wacker et al., 2002) opened up the exciting possibility to use E. coli to synthesize glycoproteins. Since this seminal work, E. coli was shown to glycosylate the native PglB (the oligosaccharide transferase from C. Jejuni) substrate, AcrA, with various O-antigen polysaccharides native to E. coli (Feldman et al., 2005;Wacker et al., 2006). More recently, attempts were made to produce glycosylated proteins other than the native PglB substrate AcrA or other types of glycans non-native to either E. coli or C. jejuni (Fisher et al., 2011;Ihssen et al., 2010;Lizak et al., 2011;Pandhal et al., 2011). Ihssen et al. used the system to attach Schigella O antigens to two carrier proteins derived from C jejuni and Pseudomonas aeruginosa, respectively. The resulting glycoproteins have the potential to be used as vaccines against shigellosis, a disease causing 1 million deaths per year in developing world, mostly of young children (Ihssen et al., 2010). In this work, the host strain CLM24 carries a chromosomal deletion of the waaL gene encoding an O polysaccharide ligase. The genes encoding the necessary components: PglB, carrier proteins, and Shiga O antigen biosynthesis were put on three compatible plasmids. Successful production of glycoproteins was detected by using the anti-shigella O1 antibodies. A ladder of glycoprotein bands were observed for each glycoprotein, due to polymerization of the repeating O-units by the action of the enzymes Wzy and Wzz. This study also identified several challenges of the glycosylation. First challenge is that glycosylation is often incomplete. In fact, glycosylation efficiency is sometimes as low as single digit percentage. When more than one glycosylation sites are present, heterogeneity is also an issue. Second challenge is the growth impairment by one or more glycosylation components and conditions for optimal production of carrier proteins may be different from those leading

Research will be limited to:
- Organism: E. coli
- Organism: Yeast
- Attribute: Protein Expression Yield
- Attribute: Post-translational Modifications (PTMs)
- Attribute: Cost
- Attribute: Timeline

Strategy: The user wants a direct comparison between E. coli and Yeast (likely S. cerevisiae and P. pastoris) across four specific metrics: yield, PTMs, cost, and timeline. I need to find quantitative or semi-quantitative data to populate a comparison table. While general differences are well-known (e.g., E. coli is faster/cheaper but lacks complex PTMs), specific benchmarks for 'yield' (mg/L) and 'timeline' (weeks/months) will provide a more professional answer. I will search for review articles or industrial white papers that provide these side-by-side metrics.
Queries:
- comparison table E. coli vs yeast protein expression yield PTMs cost timeline
- E. coli vs Pichia pastoris vs Saccharomyces cerevisiae protein expression comparison yield cost timeline PTMs
- industrial protein production cost and timeline comparison E. coli vs yeast

🔍 Reading and summarizing 5 new websites...

1. Protein Expression Host Systems | Selection Guide, Comparison ...
2. Comparison of Protein Expression Systems
3. E. coli vs. Yeast: Which Protein Expression System Is Better?
4. Strategies for Enhancing Recombinant Protein Expression
5. Protein Expression Platforms and the Challenges of Viral Antigen ...

Found 5 websites. Summarized content:

Protein Expression Host Systems | Selection Guide, Comparison ...
Okay, here is the comparison table based on the textual content provided from Beta LifeScience, according to your user research request, focusing on E. coli and Yeast:

E. coli vs. Yeast Protein Expression Comparison

Comparison of Protein Expression Systems
Here's a comparison table summarizing the protein expression systems of E. coli and Yeast based on the provided text:

E. coli vs. Yeast: Which Protein Expression System Is Better?
| Attribute | E. coli | Yeast |
| ----------------------------- | -------------------------------------------------------------------------------------------------------- | -------------------------------------------------------------------------------------------------------------------------------------------------------------- |
| Protein Expression Yield | Rapid growth allows for large quantities of protein in a short period. Can be reduced by inclusion bodies. | Slower growth compared to E. coli. Yeast tends to secrete proteins into the culture medium, simplifying purification and potentially improving yields. |
| Post-translational Modifications (PTMs) | Limited ability to perform PTMs. | Capable of performing many PTMs found in higher eukaryotes. Glycosylation patterns may differ from higher eukaryotes. |
| Cost | Cost-effective; can grow in simple, inexpensive media. | Typically more expensive than E. coli due to slower growth. |
| Timeline | Rapid growth rate, allowing for protein production in a short period. | Slower growth rate compared to E. coli, increases time associated with protein production. |

Strategies for Enhancing Recombinant Protein Expression

E. coli vs. Yeast Protein Expression - Comparison Table

Protein Expression Platforms and the Challenges of Viral Antigen ...
Okay, here is a comparison table that summarizes the relevant information from the provided website content regarding E. coli and Yeast protein expression, focusing on the constraints outlined in your request:

E. coli vs. Yeast Protein Expression for Viral Antigen Production

Key Considerations

• E. coli: Primarily suitable for smaller recombinant proteins without complex post-translational modification requirements. Inclusion body formation is a common issue, requiring refolding steps. Endotoxin contamination (LPS) is another significant consideration, requiring removal.
• Yeast: A good system for secreted proteins. The artificial glycoengineering of P. pastoris strains has allowed for the development of designer glycosylation pathways with hybrid N-glycosylation, galactosylation and sialyation complex glycans.

Please note that this table summarizes information extracted from the provided document and is limited to the details contained within. Production yields and timelines can vary substantially depending on the specific protein.