Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293F cells.     

InsectDirectTM System: Rapid, High-level Protein Expression and Purification from Insect Cells

A fundamental challenge in high-throughput (HT) expression screening is to rapidly identify the appropriate expression system for many targets in parallel. Known or unknown open reading frames (ORFs) are typically amplified by PCR and then cloned into a variety of vectors, producing recombinants used to direct target protein expression in Escherichia coli, insect cells, mammalian cells, or yeast. To facilitate rapid expression and purification in Spodoptera insect cells (Sf9), we developed transient expression vectors that include an enterokinase cleavage site immediately upstream of a ligation-independent cloning site (Ek/LIC). We also developed a high-efficiency insect cell transfection reagent, and automation-compatible fusion protein purification system for insect cells to facilitate expression screening and protein production. Positive clones identified from the small-scale screening were subjected to a larger scale production. Using this InsectDirect^TM approach, we successfully expressed milligram quantities of different human proteins including heat shock proteins, phospholipases, and protein kinases.     

Investigation of relationship between EBNA-1 expression level and specific foreign protein productivity in transient gene expression of HEK293 cells

In an attempt to determine the relationship between the Epstein–Barr virus nuclear antigen-1 (EBNA-1) expression level and specific foreign protein productivity (q_p), EBNA-1-amplifed HEK293 cells, which achieved a higher EBNA-1 expression level than that achieved by HEK293E cells, were established using dihydrofolate reductase (dhfr)-mediated gene amplification. Compared with a control culture in a null pool, Fc-fusion protein production by transient transfection in the EBNA-1-amplified pool showed a significant improvement. q_p was linearly correlated with the EBNA-1 expression level in the transient transfection of EBNA-1-amplified clones, as indicated by the correlation coefficient (R² = 0.7407). The Fc-fusion protein production and q_p in a transient gene expression-based culture with EBNA-1-amplified HEK293 cells, E-amp-68, were approximately 2.0 and 3.2 times, respectively, higher than those in a culture with HEK293E cells. The increase in q_p by EBNA-1 amplification mainly resulted from an enhancement in the amount of replicated DNA and level of mRNA expression but not an improved transfection efficiency. Taken together, it was found that EBNA-1 amplification could improve the therapeutic protein production in an HEK293 cell-based transient gene expression system.     

Large-scale transient transfection of serum-free suspension-growing HEK293 EBNA1 cells: peptone additives improve cell growth and transfection efficiency

Large-scale transient transfection of mammalian cells is a recent and powerful technology for the fast production of milligram amounts of recombinant proteins (r-proteins). As many r-proteins used for therapeutic and structural studies are naturally secreted or engineered to be secreted, a cost-effective serum-free culture medium that allows their efficient expression and purification is required. In an attempt to design such a serum-free medium, the effect of nine protein hydrolysates on cell proliferation, transfection efficiency, and volumetric productivity was evaluated using green fluorescent protein (GFP) and human placental secreted alkaline phosphate (SEAP) as reporter genes. The suspension growing, serum-free adapted HEK293SF-3F6 cell line was stably transfected with an EBNA1-expression vector to increase protein expression when using EBV oriP bearing plasmids. Compared to our standard serum-free medium, concomitant addition of the gelatin peptone N3 and removal of BSA slightly enhanced transfection efficiency and significantly increased volumetric productivity fourfold. Using the optimized medium formulation, transfection efficiencies between 40-60% were routinely obtained and SEAP production reached 18 mg/L(-1). To date, we have successfully produced and purified over fifteen r-proteins from 1-14-L bioreactors using this serum-free system. As examples, we describe the scale-up of two secreted his-tagged r-proteins Tie-2 and Neuropilin-1 extracellular domains (ED) in bioreactors. Each protein was successfully purified to >95% purity following a single immobilized metal affinity chromatography (IMAC) step. In contrast, purification of Tie-2 and Neuropilin-1 produced in serum-containing medium was much less efficient. Thus, the use of our new serum-free EBNA1 cell line with peptone-enriched serum-free medium significantly improves protein expression compared to peptone-less medium, and significantly increases their purification efficiency compared to serum-containing medium. This eliminates labor-intensive and expensive chromatographic steps, and allows for the simple, reliable, and extremely fast production of milligram amounts of r-proteins within 5 days posttransfection.     

Adding l-lysine derivatives to the genetic code of mammalian cells with engineered pyrrolysyl-tRNA synthetases

We report a method for site-specifically incorporating l-lysine derivatives into proteins in mammalian cells, based on the expression of the pyrrolysyl-tRNA synthetase (PylRS)-tRNA^Pyl pair from Methanosarcina mazei. Different types of external promoters were tested for the expression of tRNA^Pyl in Chinese hamster ovary cells. When tRNA^Pyl was expressed from a gene cluster under the control of the U6 promoter, the wild-type PylRS-tRNA^Pyl pair facilitated the most efficient incorporation of a pyrrolysine analog, N^ε-tert-butyloxycarbonyl-l-lysine (Boc-lysine), into proteins at the amber position. This PylRS-tRNA^Pyl system yielded the Boc-lysine-containing protein in an amount accounting for 1% of the total protein in human embryonic kidney (HEK) 293 cells. We also created a PylRS variant specific to N^ε-benzyloxycarbonyl-l-lysine, to incorporate this long, bulky, non-natural lysine derivative into proteins in HEK293. The recently reported variant specific to N^ε-acetyllysine was also expressed, resulting in the genetic encoding of this naturally-occurring lysine modification in mammalian cells.     

Development of a Chinese hamster ovary cell line for recombinant adenovirus-mediated gene expression

Recombinant human adenovirus (rhAd) has been used extensively for functional protein expression in mammalian cells including those of human and nonhuman origin. High-level protein production by rhAd vectors is expected in their permissive host cells, such as the human embryonic kidney 293 (HEK293) cell line. This is attributed primarily to the permissiveness of HEK293 to rhAd infection and their ability to support viral DNA replication by providing the missing El proteins. However, the HEK293 cells tend to suffer from cytopathic effect (CPE) as a result of virus replication. Under these circumstances, the host cell function is compromised and the culture viability will be reduced. Consequently, newly synthesized polypeptides may not be processed properly at posttranslational levels. Therefore, the usefulness of HEK293 cells for the expression of complex targets such as secreted proteins could be limited. In the search for a more robust cell line as a production host for rhAd expression vectors, a series of screening experiments was performed to isolate clones from Chinese hamster ovary-K1 (CHO-K1) cells. First, multiple rounds of infection of CHO-K1 cells were performed utilizing an rhAd expressing GFP. After each cycle of infection, a small population of CHO cells with high GFP levels was enriched by FACS. Second, individual clones more permissive to human adenovirus infection were isolated from the highly enriched subpopulation by serial dilution. A single clone, designated CHO-K1-C5, was found to be particularly permissive to rhAd infection than the parental pool and has served as a production host in the successful expression of several secreted proteins.     

Purification of recombinant proteins from mammalian cell culture using a generic double-affinity chromatography scheme

Transient transfection of mammalian cells has proven to be a useful technique for the rapid production of recombinant proteins because of its ability to produce milligram quantities within 2 weeks following cloning of their corresponding cDNA. This rapid production also requires a fast and efficient purification scheme that can be applied generically, typically through the use of affinity tags such as the polyhistidine-tag for capture by immobilized metal-affinity chromatography (IMAC) or the Strep-tag II, which binds to the StrepTactin affinity ligand. However, one-step purification using either of these tags has disadvantages in terms of yield, elution conditions, and purity. Here, we show that the addition of both Strep-tag-II and (His)(8) to the C-terminal of r-proteins allows efficient purification by consecutive IMAC and StrepTactin affinity. This approach has been successfully demonstrated using the intracellular protein DsRed, as well as two secreted proteins, secreted alkaline phosphatase (SEAP) and vascular endothelial growth factor (VEGF), all produced by transient transfection of HEK293-EBNA1 cells in medium supplemented with bovine calf serum. All proteins were purified to >99% homogeneity with yields varying from 29 to 81%.     

Transient Transfection Factors for High-Level Recombinant Protein Production in Suspension Cultured Mammalian Cells

The efficient transfection of cloned genes into mammalian cells system plays a critical role in the production of large quantities of recombinant proteins (r-proteins). In order to establish a simple and scaleable transient protein production system, we have used a cationic lipid-based transfection reagent-FreeStyle MAX to study transient transfection in serum-free suspension human embryonic kidney (HEK) 293 and Chinese hamster ovary (CHO) cells. We used quantification of green fluorescent protein (GFP) to monitor transfection efficiency and expression of a cloned human IgG antibody to monitor r-protein production. Parameters including transfection reagent concentration, DNA concentration, the time of complex formation, and the cell density at the time of transfection were analyzed and optimized. About 70% GFP-positive cells and 50-80 mg/l of secreted IgG antibody were obtained in both HEK-293 and CHO cells under optimal conditions. Scale-up of the transfection system to 1 l resulted in similar transfection efficiency and protein production. In addition, we evaluated production of therapeutic proteins such as human erythropoietin and human blood coagulation factor IX in both HEK-293 and CHO cells. Our results showed that the higher quantity of protein production was obtained by using optimal transient transfection conditions in serum-free adapted suspension mammalian cells.     

Integrated non-invasive system for quantifying secreted human therapeutic hIL2

Cell encapsulation has been used to treat diabetes, amyotrophic lateral sclerosis, and other chronic ailments by the secretion of therapeutic proteins in vivo. Detection of these proteins typically requires invasive procedures such as blood sampling or device extraction, however. In this article, a non-invasive means of measuring secreted protein concentration using a co-expressed red fluorescent protein marker is developed. A bicistronic expression vector was constructed for the intracellular production of a red fluorescent protein marker and the secreted production of human interleukin-2 (hIL2). The destabilized red fluorescent protein, DsExDR, was selected for its rapid turnover, as well as its ability to emit red light, which is readily transmitted through mammalian tissue. Transfections of this bicistronic vector into three cell lines C2C12, HEK293, and Jurkat showed linear correlations between the expressed proteins, DsExDR (intracellular) and hIL2 (secreted), with transfection DNA concentration. Correspondingly, there was a linear correlation between secreted product (hIL2) and intracellular marker (DsExDR). As transfection DNA was increased, Jurkat cells were found to increase secreted hIL2 in direct proportion to the accumulated DsExDR. HEK293 and C2C12 cells expressed and secreted significantly more hIL2 than the Jurkat cells, while still maintaining a linear relationship. Thus, all three cell lines were suitable hosts for the bicistronic expression of DsExDR and expression and secretion of therapeutic hIL2. This reporting strategy may find the greatest use in cell encapsulation therapy.     

A leader sequence capable of enhancing RNA expression and protein synthesis in mammalian cells

Many applications in biotechnology require human proteins generated from human cells. Stable cell lines commonly used for this purpose are difficult to develop, and scaling to large numbers of proteins can be problematic. Transient expression can circumvent this problem, but protein yields are generally too low for most applications. Here we report a novel 37‐nucleotide leader sequence that promotes rapid and high transgene expression in mammalian cells. This sequence was identified by in vitro selection and functions in a transient vaccinia‐based cytoplasmic expression system. Vectors containing this sequence produce microgram levels of protein in just 6 h from a small‐scale expression in 10⁶ cells. This level of protein synthesis is ideal for high throughput production of human proteins, and could be scaled to generate milligram quantities of protein. The technology is compatible with a broad range of cell lines, accepts plasmid and linear DNA, and functions with viruses that are approved for use under BSL1 conditions. We suggest that these advantages provide a powerful method for generating human protein in mammalian cells.     

Recombinase-mediated cassette exchange-based screening of a CRISPR/Cas9 library for enhanced recombinant protein production in human embryonic kidney cells: Improving resistance to hyperosmotic stress

Targeted engineering of mammalian cells has been widely attempted to ensure the efficient production of therapeutic proteins with proper quality during bioprocesses. However, the identification of novel targets for cell engineering is labor-intensive and has not yet been fully substantiated. Here, we established a CRISPR/Cas9 library screening platform in human embryonic kidney (HEK293) cells based on guide RNA integration mediated by recombinase-mediated cassette exchange (RMCE) to interrogate gene function in a high-throughput manner. This platform was further advanced using a nuclear localization signal-tagged recombinase that increased RMCE efficiency by 4.8-fold. Using this platform, we identified putative target genes, such as CDK8, GAS2L1, and GSPT1, and their perturbation confers resistance to hyperosmotic stress that inhibits cell growth and induces apoptosis. Knockout of these genes in monoclonal antibody (mAb)-producing recombinant HEK293 (rHEK293) cells enhanced resistance to hyperosmotic stress-induced apoptosis, resulting in enhanced mAb production. In particular, GSPT1-knockout yielded 2.3-fold increase in maximum mAb concentration in fed-batch culture where hyperosmotic stress naturally occurs due to nutrient feeding. Taken together, this streamlined screening platform allows the identification of novel targets associated with hyperosmotic stress, enabling the development of stress-resistant cells producing recombinant proteins.     

Engineered transient and stable overexpression of translation factors eIF3i and eIF3c in CHOK1 and HEK293 cells gives enhanced cell growth associated with increased c-Myc expression and increased recombinant protein synthesis

There is a desire to engineer mammalian host cell lines to improve cell growth/biomass accumulation and recombinant biopharmaceutical protein production in industrially relevant cell lines such as the CHOK1 and HEK293 cell lines. The over-expression of individual subunits of the eukaryotic translation factor eIF3 in mammalian cells has previously been shown to result in oncogenic properties being imparted on cells, including increased cell proliferation and growth and enhanced global protein synthesis rates. Here we report on the engineering of CHOK1 and HEK cells to over-express the eIF3i and eIF3c subunits of the eIF3 complex and the resultant impact on cell growth and a reporter of exogenous recombinant protein production. Transient over-expression of eIF3i in HEK293 and CHOK1 cells resulted in a modest increase in total eIF3i amounts (maximum 40% increase above control) and an approximate 10% increase in global protein synthesis rates in CHOK1 cells. Stable over-expression of eIF3i in CHOK1 cells was not achievable, most likely due to the already high levels of eIF3i in CHO cells compared to HEK293 cells, but was achieved in HEK293 cells. HEK293 cells engineered to over-express eIF3i had faster growth that was associated with increased c-Myc expression, achieved higher cell biomass and gave enhanced yields of a reporter of recombinant protein production. Whilst CHOK1 cells could not be engineered to over-express eIF3i directly, they could be engineered to over-express eIF3c, which resulted in a subsequent increase in eIF3i amounts and c-Myc expression. The CHOK1 eIF3c engineered cells grew to higher cell numbers and had enhanced cap- and IRES-dependent recombinant protein synthesis. Collectively these data show that engineering of subunits of the eIF3 complex can enhance cell growth and recombinant protein synthesis in mammalian cells in a cell specific manner that has implications for the engineering or selection of fast growing or high producing cells for production of recombinant proteins.     

Enhanced Production of Chikungunya Virus-Like Particles Using a High-pH Adapted Spodoptera frugiperda Insect Cell Line

Chikungunya virus-like particles (VLPs) have potential to be used as a prophylactic vaccine based on testing in multiple animal models and are currently being evaluated for human use in a Phase I clinical trial. The current method for producing these enveloped alphavirus VLPs by transient gene expression in mammalian cells presents challenges for scalable and robust industrial manufacturing, so the insect cell baculovirus expression vector system was evaluated as an alternative expression technology. Subsequent to recombinant baculovirus infection of Sf21 cells in standard culture media (pH 6.2–6.4), properly processed Chikungunya structural proteins were detected and assembled capsids were observed. However, an increase in culture pH to 6.6–6.8 was necessary to produce detectable concentrations of assembled VLPs. Since this elevated production pH exceeds the optimum for growth medium stability and Sf21 culture, medium modifications were made and a novel insect cell variant (SfBasic) was derived by exposure of Sf21 to elevated culture pH for a prolonged period of time. The high-pH adapted SfBasic insect cell line described herein is capable of maintaining normal cell growth into the typical mammalian cell culture pH range of 7.0–7.2 and produces 11-fold higher Chikungunya VLP yields relative to the parental Sf21 cell line. After scale-up into stirred tank bioreactors, SfBasic derived VLPs were chromatographically purified and shown to be similar in size and structure to a VLP standard derived from transient gene expression in HEK293 cells. Total serum anti-Chikungunya IgG and neutralizing titers from guinea pigs vaccinated with SfBasic derived VLPs or HEK293 derived VLPs were not significantly different with respect to production method, suggesting that this adapted insect cell line and production process could be useful for manufacturing Chikungunya VLPs for use as a vaccine. The adaptation of Sf21 to produce high levels of recombinant protein and VLPs in an elevated pH range may also have applications for other pH-sensitive protein or VLP targets.     

Comparative insight into expression of recombinant human VEGF111b,a newly identified anti-angiogenic isoform,in eukaryotic cell lines

VEGF-A is a critical growth factor in tumor growth and progression. Two families of VEGF-A isoforms are produced through alternative splicing including VEGFxxx pro-angiogenic and VEGFxxxb anti-angiogenic isoforms. VEGF111b is a new member of the VEGFxxxb family that is induced by mitomycin C and doesn't express in normal conditions. The potent anti-angiogenic properties of VEGF-111b and its remarkable resistance to proteolysis make it an interesting alternative candidate for therapeutic use in all types of cancers. Here, the recombinant VEGF-111b cDNA with insertion of intronic sequence was constructed by using a class IIs restriction enzyme-based method. The recombinant pBud-VEGF111b was transfected into CHO dhfr⁻ and HEK 293 cell lines which are currently the standard hosts for the production of candidate therapeutic proteins. Then, the VEGF-111b expression was evaluated in two cell lines using the Real-time PCR. The production of VEGF-111b protein was also investigated here by dot blotting. The VEGF expression was increased about 109 and 185-folds in transfected CHO-dhfr⁻ and HEK 293 cells, respectively, in comparison with the un-transfected cells. Dot blotting approach confirmed that both cell lines have successfully produced the VEGF-111b protein.     

Rational vector design and multi-pathway modulation of HEK 293E cells yield recombinant antibody titers exceeding 1 g/l by transient transfection under serum-free conditions

Transient transfection allows for fast production of recombinant proteins. However, the current bottlenecks in transient transfection are low titers and low specific productivity compared to stable cell lines. Here, we report an improved transient transfection protocol that yields titers exceeding 1 g/l in HEK293E cells. This was achieved by combining a new highly efficient polyethyleneimine (PEI)-based transfection protocol, optimized gene expression vectors, use of cell cycle regulators p18 and p21, acidic Fibroblast Growth Factor, exposure of cells to valproic acid and consequently the maintenance of cells at high cell densities (4 million cells/ml). This protocol was reproducibly scaled-up to a working volume of 2 l, thus delivering >1 g of purified protein just 2 weeks after transfection. This is the fastest approach to gram quantities of protein ever reported from cultivated mammalian cells and could initiate, upon further scale-up, a paradigm shift in industrial production of such proteins for any application in biotechnology.     

Uniform stable-isotope labeling in mammalian cells: formulation of a cost-effective culture medium

Uniform stable-isotope labeling of mammalian cells is achieved via a novel formulation of a serum-free cell culture medium that is based on stable-isotope-labeled autolysates and lipid extracts of various microbiological origin. Yeast autolysates allow complete replacement of individual amino acids and organic acids in a chemically defined medium (DMEM/F12), enabling a cost-effective formulation of a stable-isotope-labeled culture medium for mammalian cells. In addition, biomass-derived hydrolysates, autolysates, and lipid extracts of various classes of algae were explored as cell culture components, both separately and in combination with yeast autolysates. Optimal autolysate concentrations were established. Such novel medium formulations were tested on mammalian cell lines, often used for recombinant protein production, i.e., Chinese hamster ovary (CHO) and human embryonic kidney (HEK 293). Special attention was paid to the adaptation of these mammalian cell lines to serum-free media. Formulation of the novel proprietary cell culture medium PLIm, based on yeastolates instead of individual amino acids and organic acids, allows a four- to eightfold cost reduction for ¹⁵N and ¹³C,¹⁵N stable-isotope-labeling, respectively, in CHO cells and a three- to sixfold cost reduction in HEK 293 cells. A high level of stable-isotope enrichment of mammalian cells (>90%) was achieved within four passages by complete replacement of carbon and nitrogen sources in the medium with their stable-isotope-labeled analogs. These conditions can be used to more cost-effectively produce labeled recombinant proteins in mammalian cells.     

Optimisation of the Factor VIII yield in mammalian cell cultures by reducing the membrane bound fraction

In vivo, clotting Factor VIII (FVIII) circulates in plasma bound to von Willebrand factor (vWF), and the vWF:FVIII complex prevents binding of FVIII to phosphatidylserine (PS). Activation of FVIII by thrombin releases FVIII from vWF, and subsequently FVIII binds to PS exposed on activated platelets and forms the tenase complex together with clotting Factor IX. In vitro, during serum free production of recombinant FVIII (rFVIII), production cells also expose PS, and since vWF is not present to hinder interaction of secreted rFVIII with PS, rFVIII is partly associated with the cell membrane of the production cells. Recently, we showed that as much as 90% of secreted rFVIII is bound to transiently transfected production cells during serum free conditions. In this study, we investigated the effect of including vWF in the serum free medium, and demonstrate that addition of vWF results in release of active membrane bound rFVIII to the culture medium. Moreover, the attachment of rFVIII to cell membranes of un-transfected HEK293 cells was studied in the presence of compounds that competes for interactions between rFVIII and PS. Competitive assays between iodinated rFVIII (¹²⁵I-rFVIII) and annexin V or ortho-phospho-l-serine (OPLS) demonstrated that annexin V and OPLS were able to reduce the membrane bound fraction of rFVIII by 70% and 30%, respectively. Finally, adding OPLS to CHO cells stably expressing FVIII increased the yield by 50%. Using this new knowledge, the recovery of rFVIII could be increased considerably during serum free production of this therapeutic protein.     

基于逆转录病毒载体的外源基因表达系统的评价和应用

逆转录病毒表达系统是基因治疗研究和RNA干扰技术广泛采用的外源基因表达系统。文中以增强型绿色荧光蛋白 (EGFP) 基因的表达水平和稳定性为指标,比较逆转录病毒表达载体pQCXIN和pcDNA3.1(+) 表达质粒介导的外源基因在HEK293细胞和CHO-K1细胞的表达效率。病毒感染HEK293细胞和CHO-K1细胞的相对荧光强度 (Relative fluorescence intensity,RFI) 均约为对应的质粒转染细胞的2倍。多轮反复感染逆转录病毒表达载体能有效提高HEK293细胞表达EGFP的效率。HEK293细胞经4轮病毒感染后的RFI值较1次病毒感染HEK293细胞的RFI值约提高2倍。此外,逆转录病毒表达载体介导的外源基因表达的稳定性优于质粒转染的外源基因表达。采用携带人重组活性蛋白C (Recombinant human activated protein C,rhAPC) 基因的pQCXIN和HEK293细胞进一步验证了逆转录病毒载体介导的外源基因表达效率,构建了rhAPC表达水平为10~15 mg/(106 cells·d) 的HEK293细胞系。研究结果表明,逆转录病毒表达系统是有应用价值的介导外源基因在哺乳动物细胞高效表达的技术途径。     

Sex hormones affect the production of recombinant Factor IX in CHO and HEK-293 cell lines

Recombinant human Factor IX (rFIX) was cloned in a mammalian expression vector and transfected into CHO and HEK-293. Treatment with 10⁻⁹ M methyl testosterone increased rFIX production by 30–50% in CHO and HEK clones. However, 10⁻⁹ M 17β-oestradiol increased production of rFIX by ~50% in CHO-F7 clone and decreased production by 48% and 37% in CHO-F8 and HEK-F2-6, respectively. Progesterone treatment inhibited rFIX production in both cell lines. Production of rFIX can thus be increased by sex hormone treatment and therefore used to enhance biotechnological production in mammalian cells.