The production of recombinant human laminin-332 in a Leishmania tarentolae expression system

Laminin (LM)-332 (α3β3γ2), a large heterotrimeric glycoprotein, is an essential component of epithelial basement membranes that promotes cell adhesion and migration. Here, we expressed human LM-332 using a novel protein expression system based on the trypanosomatid protozoan host Leishmania tarentolae. Plasmids containing cDNA encoding full-length β3 and γ2 subunits and truncated α3 subunit were sequentially introduced into L. tarentolae. A recombinant strain harboring the three subunits of human LM-332 efficiently formed heterotrimer and secreted it into the culture medium. Heterotrimeric recombinant LM-332 (rLM-332) could be purified from culture medium with one-step immuno-affinity chromatography. The eluted fraction contained all three subunits, as confirmed by immunoprecipitation and immunoblotting. The purified rLM-332 showed similar cell adhesion activity to rLM-332 purified from mammalian cells, indicating its proper folding and assembly. The obtained expression level was not high; however, we suggest that this expression system has the potential for mass production of LMs for tissue engineering.     

Efficient expression and purification of recombinant human m-calpain using an Escherichia coli expression system at low temperature

Calpain belongs to the superfamily of Ca(2+)-regulated cysteine proteases, which are indispensable to the regulation of various cellular functions. Of the 15 mammalian calpain isoforms, μ- and m-calpains are the best characterized. Both μ- and m-calpain are ubiquitously expressed and exist as heterodimers, containing a distinct 80-kDa catalytic subunit (CAPN1 and CAPN2, respectively) and the common, 30-kDa regulatory subunit (CAPNS1). To date, various expression systems have been developed for producing recombinant calpains for use in structural and physiological studies, however Escherichia coli systems have proven incompatible with large-scale preparation of calpain, with the exception of rat m-calpain. Here, we have established a highly efficient method to purify active recombinant human m-calpain using an E. coli expression system at low temperature (22°C). This was achieved by co-expressing CAPN2 with a C-terminal histidine-tag, and CAPNS1, lacking the first Gly-repeated region at the N-terminal. After three sequential passes through a chromatographic column, ~5 mg of human m-calpain was homogenously purified from 1 l of E. coli culture. Proteins were stable for several months. This is the first report of efficient, large-scale purification of recombinant human m-calpain using an E. coli expression system.     

重组人尿激酶原在CHO细胞中的高效表达及其纯化

用鸡β- globin的MAR序列和人看家基因延伸因子1α(hEF-1α)的调控序列以及旱獭RNA稳定与输出序列,构建了重组人尿激酶原(recombinant human pro-urokinase,rhPro - UK)的高效表达载体,在CHO细胞中获得了rhPro - UK的高效稳定表达,rhPro - UK的表达水平达到1299 IU(以百万细胞1d的表达量计).采用阳离子交换层析、疏水层析和凝胶排阻层析的三步工艺纯化表达rhPro - UK的CHO细胞培养上清液,rhPro - UK的纯度达到98％、回收率为60％ ～70％.     

Efficient expression of secreted proteases via recombinant BacMam virus

Baculovirus vectors engineered to contain mammalian cell-active promoter elements have been described as an efficient method for transduction of a broad spectrum of human cell lines at high frequency. In the first large-scale comparative study of secreted protein production using these viral vectors, we have evaluated production of 16 recombinant enzymes--specifically, we exploited these viral vectors, termed 'BacMam' viruses, to drive expression of a panel of proteases selected from all four major mechanistic classes, including secreted, lysosomal, endosomal, and type I transmembrane proteins. To allow a generic purification strategy, coding sequences were truncated to remove transmembrane and/or subcellular retention signals before introduction, in parallel, into a C-terminally Fc-tagged BacMam transfer vector. BacMam viruses were generated and subsequently evaluated for expression of Fc-tagged protein in virus-transduced HEK-F cells. The common Fc-tag enabled single-step affinity purification of secreted recombinant protein from the culture medium. Yields were excellent, with 14 of 16 genes expressed producing 10-30 mg or more purified protein per litre of culture using standardised transduction conditions. At this level, reagent demands for a typical protease high-throughput screen (HTS) could be met from expression cultures as small as 0.1-0.5 L. Our results indicate this expression system offers a highly efficient and scaleable method for production of enzymatically-active secreted proteases and may therefore represent a novel method of protein production for other secreted enzymes with significant advantages over the diverse approaches in current use.     

Efficient expression, purification, and characterization of C-terminally tagged, recombinant human asparagine synthetase

Several lines of evidence suggest that up-regulation of asparagine synthetase (AS) in human T-cells results in metabolic changes that underpin the appearance of asparaginase-resistant forms of acute lymphoblastic leukemia (ALL). Inhibitors of human AS therefore have potential as agents for treating leukemia and tools for investigating the cellular basis of AS expression and drug-resistance. A critical problem in developing and characterizing potent inhibitors has been a lack of routine access to sufficient quantities of purified, reproducibly active human AS. We now report an efficient protocol for preparing multi-milligram quantities of C-terminally tagged, wild type human AS in a baculovirus-based expression system. The recombinant enzyme is correctly processed and exhibits high catalytic activity. Not only do these studies offer the possibility for investigating the kinetic behavior of biochemically interesting mammalian AS mutants, but such ready access to large amounts of enzyme also represents a major step in the development and characterization of inhibitors that might have clinical utility in treating asparaginase-resistant ALL.     

Efficient production of human zona pellucida-3 in a prokaryotic expression system

The zona pellucida-3 (ZP3) protein plays a pivotal role in oocyte and gamete development. We aimed to produce a recombinant ZP3 peptide using the Escherichia coli secretory system and apply it to a protein chip for detecting anti-ZP3 antibodies. The ZP3 gene was cloned into the pHOA downstream of the phoA promoter and transformed into E. coli YK537. Recombinant ZP3 was secretory expressed by decreasing the inorganic phosphate concentration. Then, rZP3 was purified and coated onto a protein chip, which was used to detect AZP3A in serum samples from 63 infertile patients. The area under the receiver operating characteristic curve was 0.934. The results, in terms of AZP3A detection, of the rZP3-coated protein chip were consistent with those of the ELISA kit. Therefore, our protein chip assay has potential for diagnosis of infertility due to AZP3A, and represents a less costly and simpler assay for clinical and research applications.     

A novel prokaryotic expression system for biosynthesis of recombinant human membrane-bound catechol-O-methyltransferase

Membrane proteins constitute 20-30% of all proteins encoded by the genome of various organisms. While large amounts of purified proteins are required for pharmaceutical and crystallization attempts, there is an unmet need for the development of novel heterologous membrane protein overexpression systems. Specifically, we tested the application of Brevibacillus choshinensis cells for the biosynthesis of human membrane bound catechol-O-methyltransferase (hMBCOMT). In terms of the upstream stage moderate to high expression was obtained for complex media formulation with a value near 45 nmol/h/mg for hMBCOMT specific activity achieved at 20 h culture with 37 °C and 250 rpm. Subsequently, the efficiency for reconstitution of hMBCOMT is markedly null in the presence of ionic detergents, such as sodium dodecyl sulphate (SDS). In general, for non-ionic and zwiterionic detergents, until a detergent critic micellar concentration (CMC) of 1.0 mM, hMBCOMT shows more biological activity at lower detergent concentrations while for detergent CMC higher than 1 mM, higher detergent concentrations seem to be ideal for hMBCOMT solubilization. Indeed, from the detergents tested, the non-ionic digitonin at 0.5% (w/v) appears to be the most suitable for hMBCOMT solubilization.     

Suppression of laminin-5 expression leads to increased motility, tumorigenicity, and invasion

Laminin-5 (Ln-5) is expressed in several human carcinomas and hypothesized to contribute to tumor invasion. To understand the role of Ln-5 in human cancers, we stably delivered small interfering RNAs (siRNAs) directed against the Ln-5 gamma2 chain into JHU-022-SCC cells (022), a non-invasive oral squamous cell carcinoma (OSCC) cell line which secretes Ln-5. Lysates from gamma2 siRNA cells (022-sigamma2) had nearly undetectable levels of the gamma2 chain while the alpha3 and beta3 subunits of Ln-5 remained unchanged compared to parental and control. In conditioned medium from 022-sigamma2 cells, the gamma2 chain and the Ln-5 heterotrimer were barely detectable, similar to an invasive OSCC cell line. Conditioned medium from 022-sigamma2 cells contained less alpha3 and beta3 subunits than both parental and control. Although the proliferation and adhesive properties of the 022-sigamma2 cells remained similar to parental and control cells, 022-sigamma2 cells showed increased detachment and a fibroblastic morphology similar to invasive cells. Moreover, migration, in vitro invasion, and in vivo tumorigenicity were enhanced in 022-sigamma2 cells. Our results suggest that the Ln-5 gamma2 chain regulates the secretion of the alpha3 and beta3 subunits. More importantly, suppression of Ln-5 results in a phenotype that is representative of invasive tumor cells.     

High yield of biologically active recombinant human amelogenin using the baculovirus expression system

The amelogenins are secreted by the ameloblast cells of developing teeth; they constitute about 90% of the enamel matrix proteins and play an important role in enamel biomineralization. Recent evidence suggests that amelogenin may also be involved in the regeneration of the periodontal tissues and that different isoforms may have cell-signalling effects. During enamel development and mineralization, the amelogenins are lost from the tissue due to sequential degradation by specific proteases, making isolation of substantial purified quantities of full-length amelogenin challenging. The aim of the present study was to express and characterize a recombinant human amelogenin protein in the eukaryotic baculovirus system in quantities sufficient for structural and functional studies. Human cDNA coding for a 175 amino acid amelogenin protein was subcloned into the pFastBac HTb vector (Invitrogen), this system adds a hexa-histidine tag and an rTEV protease cleavage site to the amino terminus of the expressed protein, enabling effective one-step purification by Ni2+-NTA affinity chromatography. The recombinant protein was expressed in Spodoptera frugiperda (Sf9) insect cells and the yield of purified his-tagged human amelogenin (rHAM+) was up to 10 mg/L culture. Recombinant human amelogenin (rHAM+) was characterized by SDS-PAGE, Western blot, ESI-TOF spectrometry, peptide mapping, and MS/MS sequencing. Production of significant amounts of pure, full-length amelogenin opened up the possibility to investigate novel functions of amelogenin. Our recent in vivo regeneration studies reveal that the rHAM+ alone could bring about regeneration of the periodontal tissues; cementum, periodontal ligament, and bone.     

Efficient expression of stable recombinant human insulin-like growth factor-1 fusion with human serum albumin in Chinese hamster ovary cells

Insulin-like growth factor-1 (IGF-1) plays a crucial role in cell development, differentiation, and metabolism, and has been a potential therapeutic agent for many diseases. Chinese hamster ovary (CHO) cells are widely used for production of recombinant therapeutic proteins, but the expression level of IGF-1 in CHO cells is very low (1,500?µg/L) and the half-life of IGF-1 in blood circulation is only 4.5?min according to previous studies. Therefore, IGF-1 was fused to long-circulating serum protein human serum albumin (HSA) and expressed in CHO cells. After 8-day fed-batch culture, the expression level of HSA–IGF-1 reached 100?mg/L. The fusion protein HSA–IGF-1 was purified with a recovery of 35% using a two-step chromatographic procedure. According to bioactivity assay, the purified HSA–IGF-1 could stimulate the proliferation of NIH3T3 cells in a dose-dependent fashion and promote the cell-cycle progression. Besides this, HSA–IGF-1 could bind to IGF-1 receptor on cell membrane and activate the intracellular PI3K/AKT signaling pathway. Our study suggested that HSA fusion technology carried out in CHO cells not only provided bioactivity in HSA–IGF-1 for further research but also offered a beneficial strategy to produce other similar cytokines in CHO cells.     

Production and purification of recombinant human interleukin-5 from yeast and baculovirus expression systems

A cDNA for human interleukin-5 (hIL-5) was created from the hIL-5 gene using site-directed mutagenesis to splice out the introns in vitro. This cDNA was expressed in yeast and baculovirus systems, utilizing in both cases an in-frame fusion to the pre sequence of the alpha-mating-type factor to direct secretion. The highest level of production was achieved from Sf9 cells using a baculovirus vector in serum-containing medium (2.7 mg/l), whereas in serum-free medium ten times less hIL-5 was produced. In the yeast system much lower levels of hIL-5 were produced (12.5 micrograms/l). Recombinant hIL-5 was purified to homogeneity from serum-free baculovirus cultures. The rhIL-5 consisted of a 30-kDa homodimer linked by disulfide bridging. The purified recombinant protein had a specific activity on murine BCL1 cells of 1.5 x 10(4) U/mg, of 3 x 10(5) U/mg in the murine eosinophil differentiation factor assay, and 2.4 x 10(7) U/mg in a human peripheral eosinophil maintenance assay.     

The beta3 chain short arm of laminin-332 (laminin-5) induces matrix assembly and cell adhesion activity of laminin-511 (laminin-10)

The basement membrane (BM) protein laminin-332 (Lm332) (laminin-5) has unique activity and structure as compared with other laminins: it strongly promotes cellular adhesion and migration, and its alpha3, beta3, and gamma2 chains are all truncated in their N-terminal regions (short arms). In the present study, we investigated the biological function of the laminin beta3 chain. When the beta3 chain short arm (beta3SA) was overexpressed in HEK293 cells (beta3SA-HEK), they deposited a large amount of beta3SA and a small amount of laminin-511 (Lm511) (laminin-10) on culture plates. Control HEK293 cells secreted Lm511 but failed to deposit it. The extracellular matrix (ECM) deposited by beta3SA-HEK cells strongly promoted cell attachment and spreading. The beta3SA-HEK ECM did not directly bind Lm511, but it stimulated control HEK293 cells to deposit Lm511 on the culture plates. Although purified beta3SA did not support cell adhesion by itself, it enhanced the cell adhesion activity of Lm511. Experiments with anti-integrin antibodies also suggested that the strong cell adhesion activity of the beta3SA-HEK ECM was derived from the synergistic action of beta3SA and Lm511. It has previously been found that beta3SA binds an unknown cell surface receptor. Taken together, the present study suggests that the short arm of the laminin beta3 chain enhances the matrix assembly of Lm511 and its cell adhesion activity by interacting with its receptor.     

Efficient expression and purification of recombinant alcohol oxidase in Pichia pastoris

In order to improve the production of alcohol oxidase (AOX), a recombinant Pichia pastoris (P. pastoris) system was constructed by transformation of the plasmid pPIC9K-AOX into P. pastoris GS115. The effects of different expression conditions on alcohol oxidase activity in the culture supernatant were investigated in the shake flask scale. The results showed that the highest extracellular activity (562 U/L) of alcohol oxidase was obtained after 56 h induction with 4% methanol at OD₆₀₀ 1.0 in the medium containing 50 g/L maltose, which is about 4.2 folds higher than previously reported. High-purity functional recombinant AOX (>90%) was purified from the culture with the Ni-NTA affinity column and Sephadex G-100 chromatographical methods, with a total recovery rate of 68.9%. Further studies showed that the purified rAOX had similar enzymatic characteristics as the native enzyme, except that the thermal stability and resistance to H₂O₂ inhibition of rAOX were significantly greater compared to the previous report. The purified rAOX was well tolerant to various water-miscible organic solvents. This efficient expression and purification process will be promising for large-scale production of rAOX as an important diagnostic enzyme for alcohol detection in many areas.     

Expression and chain assembly of human laminin-332 in an insect cell-free translation system

Laminins are a family of large heterotrimeric glycoproteins comprising alpha, beta, and gamma chains. To determine the molecular mechanisms underlying chain assembly in vitro, we expressed human laminin-332 subunits in an insect cell-free translation system. We successfully produced the beta3-gamma2 heterodimer and the alpha3-beta3-gamma2 heterotrimer of the laminin coiled-coil (LCC) domain following co-translation of each chain. The alpha3-beta3 and the alpha3-gamma2 heterodimer were not detected, suggesting that the alpha3 chain can assemble with only beta3-gamma2 heterodimer to form a heterotrimer via disulfide bonds. These results are consistent with those of a previous report indicating that laminin chain assembly proceeds through the beta-gamma heterodimer to the alpha-beta-gamma heterotrimer in vivo. We suggest that the cell-free translation system is a valid system with which to study the mechanisms underlying laminin chain assembly.     

Expression, purification, and characterization of recombinant fibulin-5 in a prokaryote expression system

Fibulin-5 is a widely expressed, integrin-binding extracellular matrix protein that mediates endothelial cell adhesion and scaffolds cells to elastic fibers. To investigate anti-angiogenesis activities and context-specific activities on responsive cells of recombinant fibulin-5 (rfibulin-5) expressed in Escherichia coli, the cDNA of fibulin-5 cloned from a human placenta cDNA library was inserted into the pET32a (+) vector to allow fibulin-5 expression as a Trx fusion protein. The fusion protein Trx-fibulin-5, expressed as insoluble inclusion bodies, was solubilized and its resulting expression level reached to 15% of the total cell protein. The Trxfibulin-5 was purified effectively by N²⁺-chelating chromatography and then identified by Western blotting analysis with an anti-His tag antibody. The purified Trx-fibulin-5 was refolded by dialysis against redox reagents, and the rfibulin-5 released from the fusion protein by enterokinase cleavage was purified using a RESOURCE RPC column. The final purified rfibulin-5 effectively inhibited angiogenesis in chicken embryos in a dose-dependent manner through a chorioallantoic membrane (CAM) assay. Additionally, rfibulin-5 potently suppressed in vitro proliferation of human umbilical vein endothelial cells, but stimulated that of human dermal fibroblasts. The expression and in vitro refolding of rfibulin-5 resulted in production of an active molecule with a yield of 2.1 mg/L.     

Overexpression of human transforming growth factor-beta1 using a recombinant CHO cell expression system

Transforming growth factor-beta1 (TGF-beta1) is secreted by most cells as a high molecular weight latent complex, which consists of latent TGF-beta1 disulfide bonded to latent TGF-beta1-binding protein (LTBP). Current recombinant expression systems yield less than 1-2 mg of the mature TGF-beta1 per liter of cell culture medium. In an effort to produce large quantities of the recombinant cytokine for structural studies, we have constructed a mammalian expression system based on a modified pcDNA3.1(+) vector with a glutamine synthetase gene inserted for gene amplification. The leader peptide of TGF-beta1 was replaced with that of rat serum albumin, and an eight-histidine tag was inserted immediately after the leader sequence to facilitate protein purification. In addition, Cys 33 of TGF-beta1, which forms a disulfide bond with LTBP, was replaced by a serine residue. The resulting expression construct produced a stable clone expressing 30 mg of mature TGF-beta1 per liter of spent medium. Purified TGF-beta1 bound with high affinity to its type II receptor with a solution dissociation constant of approximately 70 nM, and was fully active in both a Mv1Lu cell growth inhibition assay and in a PAI-1 luciferase reporter assay. Owing to similarities in the synthesis, secretion, and structure of TGF-beta family members, this recombinant expression system may also be applied to the overexpression of other TGF-beta isomers and even to members of the TGF-beta superfamily to facilitate their preparation.     

Expression and purification of recombinant human interleukin-18 protein using a yeast expression system

Interleukin-18 (IL-18) has been reported to exert significant immunoregulatory effects on inhibiting tumor growth through stimulating natural killer (NK) cell cytotoxicity and promoting production of several cytokines, including interferon-gamma (IFN-gamma) and granulocyte/macrophage colony-stimulating factor (GM-CSF). Therefore, IL-18 might serve as a potential therapeutic target for cancer treatment. However, the resource of this protein limits its availability for the clinical practice. The purpose of this study was to express and purify recombinant human (h) IL-18 protein using a yeast expression system. We reported here that hIL-18 gene was cloned into pPICZaC vector for expressing a recombinant hIL-18 protein using a yeast expression system. The recombinant hIL-18 protein was purified using centrifugal filter devices, hydrophobic chromatography, and anion exchange chromatography. The yield and purity of the recombinant hIL-18 reached 45.1% and 97.6%, respectively. This recombinant hIL-18 was shown to induce IFN-gamma production by human peripheral blood mononuclear cells (PBMCs) and enhance NK cell cytotoxicity synergistically with IL-2. Furthermore, these recombinant hIL-18-induced effects were the same as those by standard hIL-18. Therefore, the yeast expression system used in this study provides a useful method to produce large-scale of hIL-18 for the clinical application.     

Plasmid pGEX-5T: An alternative system for expression and purification of recombinant proteins

Summary A novel expression vector pGEX-5T was constructed which directs the synthesis of a fusion protein with a histidine-hexapeptide and glutathione-S-transferase at its N-terminus and the recombinant protein at its C-terminus inEscherichia coli. The designed fusion gene strategy allows the purification of soluble and insoluble recombinant proteins to homogeneity with single-step affinity chromatography using immobilized glutathione and metal chelating matrix, respectively. The principle and availability of this new expression system was respectively tested with the purification of a soluble and insoluble recombinant fusion protein containing 24 and 75 amino acids of the human thrombomodulin.