Pushing and pulling proteins into the yeast secretory pathway enhances recombinant protein secretion

Yeasts and especially Pichia pastoris (syn Komagataella spp.) are popular microbial expression systems for the production of recombinant proteins. One of the key advantages of yeast host systems is their ability to secrete the recombinant protein into the culture media. However, secretion of some recombinant proteins is less efficient. These proteins include antibody fragments such as Fabs or scFvs. We have recently identified translocation of nascent Fab fragments from the cytosol into the endoplasmic reticulum (ER) as one major bottleneck. Conceptually, this bottleneck requires engineering to increase the flux of recombinant proteins at the translocation step by pushing on the cytosolic side and pulling on the ER side. This engineering strategy is well-known in the field of metabolic engineering. To apply the push-and-pull strategy to recombinant protein secretion, we chose to modulate the cytosolic and ER Hsp70 cycles, which have a key impact on the translocation process. After identifying the relevant candidate factors of the Hsp70 cycles, we combined the push-and-pull factors in a single strain and achieved synergistic effects for antibody fragment secretion. With this concept we were able to successfully engineer strains and improve protein secretion up to 5-fold for different model protein classes. Overall, titers of more than 1.3 g/L Fab and scFv were reached in bioreactor cultivations.     

Adaptive Evolution in Producing Microtiter Cultivations Generates Genetically Stable Escherichia coli Production Hosts for Continuous Bioprocessing

The production of recombinant proteins usually reduces cell fitness and the growth rate of producing cells. The growth disadvantage favors faster-growing non-producer mutants. Therefore, continuous bioprocessing is hardly feasible in Escherichia coli due to the high escape rate. The stability of E. coli expression systems under long-term production conditions and how metabolic load triggered by recombinant gene expression influences the characteristics of mutations are investigated. Iterated fed-batch-like microbioreactor cultivations are conducted under production conditions. The easy-to-produce green fluorescent protein (GFP) and a challenging antigen-binding fragment (Fab) are used as model proteins, and BL21(DE3) and BL21^Q strains as expression hosts. In comparative whole-genome sequencing analyses, mutations that allowed cells to grow unhindered despite recombinant protein production are identified. A T7 RNA polymerase expression system is only conditionally suitable for long-term cultivation under production conditions. Mutations leading to non-producers occur in either the T7 RNA polymerase gene or the T7 promoter. The host RNA polymerase-based BL21^Q expression system remains stable in the production of GFP in long-term cultivations. For the production of Fab, mutations in lacI of the BL21^Q derivatives have positive effects on long-term stability. The results indicate that adaptive evolution carried out with genome-integrated E. coli expression systems in microtiter cultivations under industrial-relevant production conditions is an efficient strain development tool for production hosts.     

Acetate‐containing substrate mixtures improve recombinant protein secretion in Schizosaccharomyces pombe

Recombinant protein secretion in yeasts poses a burden to the metabolism of the host cell. Consequently, unfavorable cultivation conditions during strain screening or process development can lead to limitations in the energy and carbon metabolism of the cell, constraining the cell's ability to secrete the protein of interest. Recently, we demonstrated that improving cultivation conditions by using substrate mixtures of glycerol and acetate strongly elevated secretion of the homologous model protein maltase in the fission yeast Schizosaccharomyces pombe. In this work, we investigated if these previous findings were also applicable to the expression of recombinant proteins. Strains were constructed secreting either green fluorescent protein or a fluorescent single‐chain antibody fragment. These strains were cultivated under fermentative and respiratory growth conditions on glucose as sole carbon source and on mixtures of glucose/acetate and glycerol/acetate. We observed an increase in the specific secretion of both recombinant proteins by 1.8‐ and 3.8‐fold, respectively. This clearly demonstrates that the proper choice of process conditions and the applied carbon sources have a significant impact on the secretion of at least two recombinant proteins in S. pombe allowing an improved production of the protein of interest.     

Biomedicals from a soil bug: expanding scFv production host range

Recombinant antibody fragments have a wide range of applications from research to diagnostics and therapy. Of special interest are small fragments like fragment antigen binding (Fab) or single chain fragment variables (scFv) fragments as they can be produced inexpensively in bacterial expression systems. However, recombinant production efficiencies from established production hosts vary significantly leading to inadequate yields. Gene sequences that have been synthetically adapted to match the codon preferences and respective genomic tRNA pool of the host have been used to improve yields but cannot resolve the principal problem. The development of inducible broad host range scFv expression plasmid constructs leads the way to an easy and efficient screening method for the identification of the optimal bacterial expression host.     

High level expression of a promising anti-idiotypic antibody fragment vaccine against HIV-1 in Pichia pastoris

We have expressed the anti-idiotypic antibody 3H6 Fab directed against the HIV-1 broadly neutralising antibody 2F5 in methylotrophic yeast Pichia pastoris. The chimeric human/mouse Fab fragment was expressed under control of the inducible AOX1 promoter and secreted via the alpha mating factor leader of Saccharomyces cerevisiae. Bioreactor experiments showed the ability of the recombinant P. pastoris clone to secrete up to 260 mg/L Fab fragment in the culture supernatant during a five days cultivation time. Codon optimisation of the Fab expression cassette gave no further improvement of specific productivity when comparing 12 clones of each construct. The subsequent purification of Fab containing supernatants was done by anion exchange and size-exclusion chromatography with a recovery resulting in 70% of the recombinant protein. For verification of the suitability of the expression system we characterised the expressed protein with respect to both, its specificity and binding affinity and could not detect any significant difference between products from yeast derived and the hybridoma derived product. Finally we tested the implicit requirement of the carbohydrate moiety in the H2 loop of the original 3H6 antibody by introducing an asparagine to alanine replacement and, in a second experiment, inhibition of N-glycosylation by tunicamycin treatment. Biochemical analysis confirmed that the N-glycosylation does not contribute to the binding properties of 3H6.     

Intracellular interactome of secreted antibody Fab fragment in <Emphasis Type="Italic">Pichia pastoris</Emphasis> reveals its routes of secretion and degradation

Protein translation, translocation, folding, processing, and secretion in eukaryotic cells are complex and not always straightforward processes, e.g., different routes of secretion and degradation exist. Formation of malfolded proteins in the endoplasmic reticulum (ER) can be one of the major bottlenecks for recombinant protein production. In this regard, an in-depth analysis of the interactions of a secreted protein during its pathway through the cell may be beneficial, as realized in this study for the methylotrophic yeast Pichia pastoris. The antibody fragment Fab3H6 used here is the anti-idiotype to the HIV neutralizing antibody 2F5 and is known to be intracellularly degraded in significant amounts when expressed in P. pastoris. The interactome of Fab3H6 was analyzed by using a pull-down mass spectrometry approach, and 23 proteins were found to bind specifically to the antibody fragment. Those allowed concluding that Fab3H6 is post-translationally translocated into the ER and degraded via the proteasome as well as the vacuole. In line with this, the expression of Fab3H6 increased the proteasomal activities by over 20%. Partial inhibition of the proteasome resulted in a significant increase of extracellular Fab3H6. Thus, it seems that ER quality control overshoots its requirements for the recombinant protein expressed and that more than just terminally malfolded protein is degraded by ER-associated degradation. This work will further facilitate our understanding how recombinant proteins behave in the secretory pathway.     

Recombinant Fab expression and secretion in Escherichia coli continuous culture at medium cell densities: Influence of temperature

Production of recombinant antibody fragments (Fabs) in Escherichia coli has gained interest because of the recognised advantages of this expression system and because Fabs do not require glycosylation. However, more comprehensive studies on the factors that influence expression conditions and product yield are still required for full process development. In this work, the effect of growth temperature on the periplasmatic expression of the 3H6 Fab in E. coli was studied in carbon-limited continuous cultures operated at medium cell densities. Three different temperatures were assayed, namely 37, 33 and 30 °C. Results showed that biomass yield was not affected within this temperature range whilst product yield increased as temperature decreased. Periplasmic Fab secretion corresponded to 30% of the produced Fab protein and its efficiency was irrespective of the process temperature. Moreover, considerable product leakage to the culture supernatant was detected in all cases, ranging from about 40% at 37 °C to almost 70% at 30 °C. Besides, plasmid loss was observed along process time indicating a selective pressure against plasmid-bearing cells. This study supports the potential of continuous cultivations of E. coli at medium cell densities under well controlled conditions as a tool for characterising the impact of environmental parameters and cell physiology under protein production conditions.     

Production of recombinant proteins by filamentous fungi

The initial focus of recombinant protein production by filamentous fungi related to exploiting the extraordinary extracellular enzyme synthesis and secretion machinery of industrial strains, including Aspergillus, Trichoderma, Penicillium and Rhizopus species, was to produce single recombinant protein products. An early recognized disadvantage of filamentous fungi as hosts of recombinant proteins was their common ability to produce homologous proteases which could degrade the heterologous protein product and strategies to prevent proteolysis have met with some limited success. It was also recognized that the protein glycosylation patterns in filamentous fungi and in mammals were quite different, such that filamentous fungi are likely not to be the most suitable microbial hosts for production of recombinant human glycoproteins for therapeutic use. By combining the experience gained from production of single recombinant proteins with new scientific information being generated through genomics and proteomics research, biotechnologists are now poised to extend the biomanufacturing capabilities of recombinant filamentous fungi by enabling them to express genes encoding multiple proteins, including, for example, new biosynthetic pathways for production of new primary or secondary metabolites. It is recognized that filamentous fungi, most species of which have not yet been isolated, represent an enormously diverse source of novel biosynthetic pathways, and that the natural fungal host harboring a valuable biosynthesis pathway may often not be the most suitable organism for biomanufacture purposes. Hence it is expected that substantial effort will be directed to transforming other fungal hosts, non-fungal microbial hosts and indeed non microbial hosts to express some of these novel biosynthetic pathways. But future applications of recombinant expression of proteins will not be confined to biomanufacturing. Opportunities to exploit recombinant technology to unravel the causes of the deleterious impacts of fungi, for example as human, mammalian and plant pathogens, and then to bring forward solutions, is expected to represent a very important future focus of fungal recombinant protein technology.     

Growth at sub-optimal temperatures allows the production of functional, antigen-binding Fab fragments in Escherichia coli

Expression in Escherichia coli of recombinant genes coding for the kappa-chain and the Fd fragment of an antibody directed against carcinoembryonic antigen gives rise to Fab dimers. These Fab fragments possess antibody activity, as demonstrated by enzyme-linked immunosorbent assay as well as by ligand competition assay. Effective production of soluble Fab in Escherichia coli was achieved by a decrease in the growth temperature. Following a one step purification by anion exchange chromatography, the bacterially-produced Fab retains its activity at 4 degrees C for at least two months. The relatively simple methodology described in this study should be useful for the design and production of antibodies in bacteria.     

Efficient bacterial production of functional antibody fragments using a phagemid vector

The so-called ‘in vitro evolutionary method’ using a phage display system has been applied for protein engineering of the antigen-binding fragment of antibodies (Fab) by conducting random mutagenesis at the antigen-binding site in combination with antigen-based biopanning. However, isolated phage clones displaying Fab cannot necessarily be used for efficient bacterial production of engineered Fab proteins, often due to deleterious defects in their proper folding abilities derived in compensation for the gain of high affinity for a particular antigen. We here report a new method of an efficient and direct bacterial expression system for the phagemid-coded Fab proteins without use of the helper phage. To overcome a low folding efficiency derived from somatic hypermutations, if any, we have established optimum conditions for bacterial cultivation and protein expression, utilizing unusually long cultivation time (>50 h) and very low temperature (25 °C) and thereby leading to the production and extracellular secretion of Fab proteins in a very high yield (3–15 mg/L of culture). The purified Fab folded correctly and could efficiently bind an antigen, as judged by circular dichroism and isothermal titration calorimetry, respectively.     

Production of a recombinant antibody fragment in whole insect larvae

Infection of insect cells with baculovirus expression constructs is commonly used to produce recombinant proteins that require post-translational modifications for their activity, such as mammalian proteins. However, technical restraints limit the capacity of insect cell-based culture systems to be scaled up to produce the large amounts of recombinant protein required for human pharmaceuticals. In this study, we designed an automated insect rearing system and whole insect baculovirus expression system (PERLXpress™) for the expression and purification of recombinant proteins on a large scale. As a test model, we produced a recombinant mouse anti-botulinum antibody fragment (Fab) in Trichoplusia ni larvae. A recombinant baculovirus co-expressing the Fab heavy and light chains together with N-terminal sequences from the silkworm hormone bombyxin, to direct proteins into the secretory pathway, was constructed. Fifth instar larvae were reared and infected orally with recombinant (pre- occluded) baculovirus using the automated system and harvested approximately after 4 days. The total yield of recombinant Fab was 1.1 g/kg of larvae, resulting in 127 mg of pure Fab in one production run. The Fab was purified to homogeneity using immobilized metal affinity chromatography, gel filtration, and anion exchange chromatography. The identity of the purified protein was verified by Western blots and size-exclusion chromatography. Purified recombinant Fab was used to detect botulinum toxin in ELISA experiments, demonstrating that the heavy and light chains were properly assembled and folded into functional heterodimers. We believe that this is the first demonstration of the expression of a recombinant antibody in whole insect larvae. Our results demonstrate that a baculovirus-whole larvae expression system can be used to express functionally active recombinant Fab fragments. As the PERLXpress™ system is an automated and linearly scalable technology, it represents an attractive alternative to insect cell culture for the production of large amounts of human pharmaceuticals.     

Neutralizing chimeric mouse-human antibodies against Burkholderia pseudomallei protease: expression, purification and characterization

A recombinant Fab monoclonal antibody (Fab) C37, previously obtained by phage display and biopanning of a random antibody fragment library against Burkholderia pseudomallei protease, was expressed in different strains of Escherichia coli. E. coli strain HB2151 was deemed a more suitable host for Fab expression than other E. coli strains when grown in media supplemented with 0.2 % glycerol. The expressed Fab fragment was purified by affinity chromatography on a Protein G-Sepharose column, and the specificity of the recombinant Fab C37 towards B. pseudomallei protease was proven by Western blotting, enzyme-linked immunosorbent assay (ELISA) and by proteolytic activity neutralization. In addition, polyclonal antibodies against B. pseudomallei protease were produced in rabbits immunized with the protease. These were isolated from high titer serum by affinity chromatography on recombinant-Protein A-Sepharose. Purified polyclonal antibody specificity towards B. pseudomallei protease was proven by Western blotting and ELISA.     

Helix-stabilized Fv (hsFv) antibody fragments: substituting the constant domains of a Fab fragment for a heterodimeric coiled-coil domain.

Antibody Fv fragments would in principle be useful for a variety of biotechnological applications because of their small size and the possibility to produce them in relatively large amounts in recombinant form; however, their limited stability is a drawback. To solve this problem, both domains are usually fused via a peptide linker to form a single-chain Fv (scFv) fragment, but in some cases this leads to a dimerization. We present an alternative format for stabilizing antibody Fv fragments. The C(H)1 and C(L) domain of the Fab fragment were replaced with a heterodimeric coiled coil (WinZip-A2B1), which had previously been selected using a protein-fragment complementation assay in Escherichia coli. This new antibody format was termed helix-stabilized Fv fragment (hsFv), and was compared to the corresponding Fv, Fab and single-chain Fv format. Bacterial growth and expression of the hsFv was significantly improved compared to the Fab fragment. The hsFv fragment formed a heterodimer of heavy and light chain with the expected molecular mass, also under conditions where the scFv fragment was predominantly dimeric. The hsFv fragment was significantly more stable than the Fv fragment, and nearly as stable as the scFv fragment under the conditions used (80 nM protein concentration). Thus, the format of a helix-stabilized Fv (hsFv) fragment can be a useful alternative to existing recombinant antibody formats, especially in cases where poor expression of Fab fragments or multimerization of scFv fragments is a problem.     

Microbioreactor Cultivations of Fab‐Producing Escherichia coli Reveal Genome‐Integrated Systems as Suitable for Prospective Studies on Direct Fab Expression Effects

Despite efforts to develop concepts for efficient antibody fragment (Fab) production in Escherichia coli (E. coli) and the high degree of similarity within this protein class, a generic platform technology is still not available. Indeed, feasible production of new Fab candidates remains challenging. In this study, a setup that enables direct characterization of host cell response to Fab expression by utilizing genome‐integrated (GI) systems is established. Among the multitude of factors that influence Fab expression, the variable domain, the translocation mechanism, the host strain, as well as the copy number of the gene of interest (GOI) are varied. The resulting 32 production clones are characterized in carbon‐limited microbioreactor cultivations with yields of 0–7.4 mg Fab per gram of cell dry mass. Antigen‐binding region variations have the greatest effect on Fab yield. In most cases, the E. coli HMS174(DE3) strain performs better than the BL21(DE3) strain. Translocation mechanism variations mainly influence leader peptide cleavage efficiency. Plasmid‐free systems, with a single copy of the GOI integrated into the chromosome, reach Fab yields in the range of 80–300% of plasmid‐based counterparts. Consequently, the GI Fab production clones could greatly facilitate direct analyses of systems response to different impact factors under varying production conditions.     

Increased heterologous protein production by Saccharomyces cerevisiae growing on ethanol as sole carbon source

Saccharomyces cerevisiae is a widely used host organism for the production of heterologous proteins, often cultivated in glucose-based fed-batch processes. This production system however has many factors limiting the productivity, mainly towards the end of the fermentation. For the optimised production of a Camelid antibody fragment this process was evaluated. In shake flask cultivations, it was found that ethanol has a strong effect on productivity increase and therefore glucose and ethanol fed-batch fermentations were compared. It appeared that specific heterologous protein production was up to five times higher in the ethanol cultivation and could be further optimised. Then the key characteristics of ethanol fed-batch fermentations such as growth rate and specific production were determined under ethanol limitation and accumulation and growth limiting conditions in the final phase of the process. It appeared that an optimal production process should have an ethanol accumulation throughout the feed phase of approximately 1% v/v in the broth and that production remains very efficient even in the last phase of the process. This productivity increase on ethanol versus glucose was also proven for several other Camelid antibody fragments some of which were heavily impaired in secretion on glucose, but very well produced on ethanol. This leads to the suggestion that the ethanol effect on improved heterologous protein production is linked to a stress response and folding and secretion efficiency.     

Production of a recombinant Fab in Pichia pastoris from a Monocistronic expression vector

Recombinant Fab is usually expressed using dicistronic vectors producing the heavy and light chains separately. We developed an improved vector for Fab fragment expression in Pichia pastoris, which allows a stoichiometric expression of both chains based on a monocistronic arrangement. The protein is produced as a unique polypeptide harbouring a KEX2 processing site between both chains. After KEX cleavage, a correctly folded mature Fab is formed. The produced recombinant protein is characterized as a heterodimeric functional Fab. The vector described is a new tool for the proper expression of antibody fragments or any heterodimeric polypeptides.     

Heterologous protein production in Zygosaccharomyces bailii: physiological effects and fermentative strategies

The optimisation and scale-up of a specific protein production process have to take into account cultivation conditions as well as cell physiology of growth and the influence of foreign protein expression on host cell metabolism. The ability of Zygosaccharomyces bailii to tolerate high sugar concentrations as well as high temperatures and acidic environments renders this "non-conventional" yeast suitable for the development of biotechnological processes like heterologous protein production. This work addresses the production of human interleukin-1beta by a recombinant Z. bailii strain. We found that the heterologous protein production causes some modifications of the Z. bailii carbon metabolism, leading to a reduced biomass yield. The other important factor is the dependence of the recombinant IL-1beta production/secretion on the growth rate. Among the cultivation strategies studied, the most appropriate in terms of production and productivity was the fed-batch mode.     

Optimisation of contained Nicotiana tabacum cultivation for the production of recombinant protein pharmaceuticals

Nicotiana tabacum is emerging as a crop of choice for production of recombinant protein pharmaceuticals. Although there is significant commercial expertise in tobacco farming, different cultivation practices are likely to be needed when the objective is to optimise protein expression, yield and extraction, rather than the traditional focus on biomass and alkaloid production. Moreover, pharmaceutical transgenic tobacco plants are likely to be grown initially within a controlled environment, the parameters for which have yet to be established. Here, the growth characteristics and functional recombinant protein yields for two separate transgenic tobacco plant lines were investigated. The impacts of temperature, day-length, compost nitrogen content, radiation and plant density were examined. Temperature was the only environmental variable to affect IgG concentration in the plants, with higher yields observed in plants grown at lower temperature. In contrast, temperature, supplementary radiation and plant density all affected the total soluble protein yield in the same plants. Transgenic plants expressing a second recombinant protein (cyanovirin-N) responded differently to IgG transgenic plants to elevated temperature, with an increase in cyanovirin-N concentration, although the effect of the environmental variables on total soluble protein yields was the same as the IgG plants. Planting density and radiation levels were important factors affecting variability of the two recombinant protein yields in transgenic plants. Phenotypic differences were observed between the two transgenic plant lines and non-transformed N. tabacum, but the effect of different growing conditions was consistent between the three lines. Temperature, day length, radiation intensity and planting density all had a significant impact on biomass production. Taken together, the data suggest that recombinant protein yield is not affected substantially by environmental factors other than growth temperature. Overall productivity is therefore correlated to biomass production, although other factors such as purification burden, extractability protein stability and quality also need to be considered in the optimal design of cultivation conditions.