Glycosyl-phosphatidylinositol-anchor addition signals are processed in Nicotiana tabacum

Recent studies have demonstrated the existence of glycosyl-phosphatidylinositol (GPI)-anchored proteins in higher plants. In this study we tested whether GPI-addition signals from diverse evolutionary sources would function to link a GPI-anchor to a reporter protein in plant cells. Tobacco protoplasts were transiently transfected with a truncated form of the Clostridium thermocellum endoglucanase E reporter gene (celE') fused with a tobacco secretion signal (PR-1a) at the N-terminus and either a yeast (GAS1), mammalian (Thy-1) or putative plant (LeAGP-1) GPI-anchor addition signal at the C-terminus. The yeast and plant C-terminal signals were found to be capable of directing the addition of a GPI-anchor to the endoglucanase protein (EGE') as shown by the sensitivity of the lipid component of GPI to phosphatidylinositol-specific phospholipase C (PI-PLC) digestion. In contrast, the mammalian signal was poorly processed for anchor addition. When EGE' was fused to a truncated form of the LeAGP-1 signal (missing three amino acids predicted to be critical to signal cleavage and anchor addition), a GPI-anchor was not linked to the EGE' protein indicating the necessity for the missing amino acids. Our results show the conservation of the properties of GPI-signals in plant cells and that there may be some similar preferences in GPI-addition signal sequences for yeast and plant cells.     

A truncated bacterial xylanase expressed in mammalian cells exhibits increased sensitivity to proteases

A truncated xylanase gene from the ruminal bacterium Fibrobacter succinogenes S85 was expressed in Chinese hamster ovary (CHO) cells and pancreatic acinar 266–6 cells using the SV40 early promoter and the mouse Amy-2.2 promoter/enhancer, respectively. The enzyme produced in both systems was active and secreted into the medium. The xylanase secreted from CHO cells was highly glycosylated and became sensitive to protease digestion compared to the unglycosylated form. © Rapid Science Ltd. 1998     

SV40 intron,a potent strong intron element that effectively increases transgene expression in transfected Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells have become the most widely utilized mammalian cell line for the production of recombinant proteins. However, the product yield and transgene instability need to be further increased and solved. In this study, we investigated the effect of five different introns on transgene expression in CHO cells. hCMV intron A, adenovirus tripartite leader sequence intron, SV40 intron, Chinese hamster EF‐1alpha gene intron 1 and intervening sequence intron were cloned downstream of the eGFP expression cassette in a eukaryotic vector, which was then transfected into CHO cells. qRT‐PCR and flow cytometry were used to explore eGFP expression levels. And gene copy number was also detected by qPCR, respectively. Furthermore, the erythropoietin (EPO) protein was used to test the selected more strong intron. The results showed that SV40 intron exhibited the highest transgene expression level among the five compared intron elements under transient and stable transfections. In addition, the SV40 intron element can increase the ratio of positive colonies and decrease the coefficient of variation in transgene expression level. Moreover, the transgene expression level was not related to the gene copy number in stable transfected CHO cells. Also, the SV40 intron induced higher level of EPO expression than IVS intron in transfected CHO cell. In conclusion, SV40 intron is a potent strong intron element that increases transgene expression, which can readily be used to more efficient transgenic protein production in CHO cells.     

人IFN-β启动子基因报告质粒的构建及SARS-CoV3a和7a蛋白对IFN-β诱生作用的初步研究

3a蛋白和7a蛋白是SARS-CoV的非结构蛋白,分别主要由SARS基因组中ORF 3a 和ORF 7a所编码。在体内和体外感染病毒的细胞中均发现了有3a蛋白的表达。首先将pGL3-Control载体进行了改构,除去了SV40启动子基因,获得了pGL3-Enhancer载体,将获得的IFN-β启动子基因连入载体中,构建了带有人IFN-β启动子基因的荧光素酶报告质粒IP-21,并且通过实验证明所构建的质粒在干扰素的诱导剂NDV的作用下能够表达荧光素酶活性,照度计检测荧光素酶活性增强,从而验证了所构建的重组质粒的有效性。为了观察SARS-CoV非结构蛋白3a和7a对干扰素诱生途径的影响,将IP-21瞬转入稳定表达有3a和7a蛋白的CHO细胞,通过荧光素酶的信号强弱对3a和7a的作用进行分析,结果表明SARS-CoV的3a和7a蛋白能够刺激荧光素酶的表达,即在体外的细胞实验中,3a和7a能有效地激活IFN-β的启动子部分。此结果对进一步研究3a和7a的功能以及对SARS-CoV的致病机制的进一步研究有一定意义。     

Expression vector system based on the chicken beta-actin promoter directs efficient production of interleukin-5

We examined the promoter activity of the 1.3-kb chicken beta-actin gene sequence located between the 5' flanking region and the proximal region of the second exon. This promoter region showed higher promoter activity than the simian virus 40 (SV40) early promoter or the Rous sarcoma virus (RSV) long terminal repeat (LTR) as assayed by transient lacZ gene expression in mouse L cells. Furthermore, replacement of the 3' splice sequence in this promoter by that derived from the rabbit beta-globin gene resulted in a approximately 2.5-fold enhancement in the synthesis of beta-galactosidase (beta Gal). Introduction of the SV40 origin of DNA replication (ori) into the vector carrying this hybrid promoter, which we designate the AG promoter, markedly enhanced the production of beta Gal in an SV40 T antigen-producing cell, BMT10. We have constructed a useful vector containing the strong AG promoter, several unique restriction sites, a SV40 polyadenylation signal and the SV40 ori for transient expression of cDNA in BMT10 or COS cells. We demonstrate the use of this vector for efficient production of interleukin-5 in BMT10 cells.     

Generating stable chinese hamster ovary cell clones to produce a truncated SARS‐CoV spike protein for vaccine development

The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS‐CoV) is important for vaccine development. S_TR2 (an 88 kDa truncated SARS‐CoV TW1 S protein carrying the S fragments S‐74‐253, S‐294‐739, and S‐1129‐1255) is capable of expressing a major form of glycoprotein as endo H‐sensitive (～115 kDa) in CHO cells. To establish stable expressing cell clones, we transfected CHO/dhFr‐cells with the amplifiable vectors ISID (IRES‐driven dhfr) and ISIZ (SV40‐driven dhfr) to select stepwise MTX, and observed enhanced ～115 kDa glycoform generation through gene amplification. Following stepwise MTX selection, we compared gene amplification levels between two vectors in engineered CHO cell chromosomes. These results confirm that the IRES‐driven dhfr promoter generates greater gene amplification, which in turn enhances S_TR2 expression. Our results indicate that the ～115 kDa glycoform of S_TR2 protein was capable of increasing after gene amplification. The S_TR2 glycoform did not change between suspension and serum‐free cultures, suggesting that the stable and amplified cell clones analyzed in this study have potential for producing homologous S_TR2 on a large scale. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Purification and characterization of recombinant human testis angiotensin-converting enzyme expressed in Chinese hamster ovary cells.

Enzymatically active human testis angiotensin-converting enzyme (ACE) was expressed in Chinese hamster ovary (CHO) cells stably transfected with each of three vectors: p omega-ACE contains a full-length testis ACE cDNA under the control of a retroviral promoter; and pLEN-ACEVII and pLEN-ACE6/5, in which full-length and membrane anchor-minus testis ACE cDNAs, respectively, are under the control of the human metallothionein IIA promoter and SV40 enhancer. In every case, active recombinant human testis ACE (hTACE) was secreted in a soluble form into the culture media, up to 2.4 mg/liter in the media of metal-induced, high-producing clones transfected with one of the pLEN vectors. In addition, membrane-bound recombinant enzyme was recovered from detergent extracts of cell pellets of CHO cells transfected with either p omega-ACE or pLEN-ACE-VII. Recombinant converting enzyme was purified to homogeneity by single-step affinity chromatography of conditioned media and detergent-extracted cell pellets in 85 and 70% overall yield, respectively. Purified hTACE from all sources comigrated with the native testis isozyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with M(r) approximately 100 kDa. The native and recombinant proteins cross-reacted equally with anti-human kidney ACE antiserum on Western blotting. The catalytic activity of recombinant angiotensin-converting enzyme, in terms of angiotensin I and 2-furanacryloyl-Phe-Gly-Gly hydrolysis, chloride activation, and lisinopril inhibition, was essentially identical to that of the native enzyme. The facile recovery in high yield of fully active hTACE from the media of stably transfected CHO cells provides a suitable system for investigating structure-function relationships in this enzyme.     

High-level expression of the bovine growth hormone gene in heterologous mammalian cells

The gene coding for bovine growth hormone (bGH) was isolated from a lambda-phage library constructed using bovine pituitary DNA partially digested with MboI. Expression of this gene transfected into mouse and monkey cells was studied. CV-1 monkey cells transfected with simian virus 40 (SV40) vectors containing the intact bGH gene, including the putative promoter region, did not express bGH. However, replacement of the bGH promoter with the mouse metallothionein-I (MT) promoter resulted in high-level synthesis and secretion of bGH. These results show that the bGH promoter functions poorly in CV-1 cells but CV-1 cells process and translate the bGH mRNA accurately. The MT-bGH chimeric gene was used to establish permanent bGH-secreting mouse C127 cell lines using the 69% transforming fragment of bovine papilloma virus (BPV) as the vector. One such cell line produced high levels of bGH and secreted it into the medium efficiently. Secreted bGH is processed accurately and is bioactive as judged by its ability to bind to rabbit liver membrane preparations.     

Human interferon omega 1: isolation of the gene, expression in Chinese hamster ovary cells and characterization of the recombinant protein.

A gene encoding human interferon omega-1 (IFN-omega 1) was isolated from a cosmid library, sequenced and expressed in Chinese hamster ovary (CHO) cells under the control of an SV40-derived promoter/enhancer sequence. Culture supernatants of stably transfected cell clones contained biologically active IFN-omega 1 at concentrations up to 10 micrograms/l. Amplification of the expression vector containing a dhfr gene under methotrexate selection pressure resulted in yields up to 200 micrograms/l. Production of IFN-omega 1 was further enhanced 2- to 3-fold by propagation of the cells in the presence of n-butyrate. IFN-omega 1 was purified from culture supernatants by monoclonal antibody affinity chromatography. The resulting protein was at least 95% pure as determined by reverse-phase HPLC and size-exclusion HPLC. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed two bands of about the same intensity with apparent molecular masses of 24.5 and 22.5 kDa. Upon treatment with peptide:N-glycosidase F, both bands were shifted to lower molecular masses (20.5 and 18.5 kDa), indicating that CHO cell-derived IFN-omega 1 is glycosylated; Asn-78 was identified as the glycosylation site. Analysis of the carbohydrate moiety using glycosidases and lectins revealed the presence of biantennary complex oligosaccharides containing neuraminic acid. Amino acid sequencing showed that only about 40% of the molecules have the expected N-terminus, whereas the others carry two additional amino acids derived from the signal sequence. C-terminal amino acid sequencing using carboxypeptidase P demonstrated that the smaller form of the protein lacks nine amino acids. Disulfide bridges were shown to connect Cys residues 1 and 99 as well as 29 and 139, respectively, as in IFN-alpha. The specific antiviral activity of recombinant, glycosylated human IFN-omega 1 on human cells was 2.6 x 10(8) IU/mg, not significantly different from that of the authentic, human leukocyte-derived protein.     

Sequences from polyomavirus and simian virus 40 large T genes capable of immortalizing primary rat embryo fibroblasts.

We developed a procedure to evaluate quantitatively the capacity of subgenomic fragments from polyomavirus and simian virus 40 (SV40) to promote the establishment of primary cells in culture. The large T antigen from both of these viruses can immortalize primary rat embryo fibroblasts. Both antigens have amino-terminal domains that retain biological activity after deletion of other parts of the polypeptide chain. However, this activity varies considerably among various mutants, presumably because of alterations in the stability or conformation of the truncated polypeptides. The polyomavirus middle T gene alone immortalizes at a low efficiency, which indicates that this oncogene can have both immortalization and transformation potentials depending on the assay system chosen. We generated deletions in the polyomavirus and SV40 large T genes to localize more precisely the functional domains of the proteins involved in the immortalization process. Our results show that the region of the SV40 large T antigen involved in immortalization is localized within the first 137 amino acid residues. This region is encoded by the first large T exon and a small portion from the second exon which includes the SV40 large T nuclear location signal. The polyomavirus sequence involved in immortalization comprises a region from the second large T exon, mapping between nucleotides 1016 and 1213, which shares no homology with SV40 and is thought to be of cellular origin. We suggest that this region of the polyomavirus large T gene functions either as a nuclear location signal or as part of the large T protein sequence involved in DNA binding.     

Identification of the erythropoietin receptor domain required for calcium channel activation.

Erythropoietin (Epo) activates a voltage-independent Ca2+ channel that is dependent on tyrosine phosphorylation. To identify the domain(s) of the Epo receptor (Epo-R) required for Epo-induced Ca2+ influx, Chinese hamster ovary (CHO) cells were transfected with wild-type or mutant Epo receptors subcloned into pTracer-cytomegalovirus vector. This vector contains an SV40 early promoter, which drives expression of the green fluorescent protein (GFP) gene, and a cytomegalovirus immediate-early promoter driving expression of the Epo-R. Successful transfection was verified in single cells by detection of GFP, and intracellular Ca2+ ([Ca]i) changes were simultaneously monitored with rhod-2. Transfection of CHO cells with pTracer encoding wild-type Epo-R, but not pTracer alone, resulted in an Epo-induced [Ca]i increase that was abolished in cells transfected with Epo-R F8 (all eight cytoplasmic tyrosines substituted). Transfection with carboxyl-terminal deletion mutants indicated that removal of the terminal four tyrosine phosphorylation sites, but not the tyrosine at position 479, abolished Epo-induced [Ca]i increase, suggesting that tyrosines at positions 443, 460, and/or 464 are important. In CHO cells transfected with mutant Epo-R in which phenylalanine was substituted for individual tyrosines, a significant increase in [Ca]i was observed with mutants Epo-R Y443F and Epo-R Y464F. The rise in [Ca]i was abolished in cells transfected with Epo-R Y460F. Results were confirmed with CHO cells transfected with plasmids expressing Epo-R mutants in which individual tyrosines were added back to Epo-R F8 and in stably transfected Ba/F3 cells. These results demonstrate a critical role for the Epo-R cytoplasmic tyrosine 460 in Epo-stimulated Ca2+ influx.     

Generation of a biologically active, secreted form of human thyroid peroxidase by site-directed mutagenesis

Using site-directed mutagenesis, we introduced two stop codons immediately upstream of the putative transmembrane domain in human thyroid peroxidase (hTPO) cDNA, truncating the carboxyl terminus of hTPO (933 amino acids) by 85 residues. Mutated hTPO cDNA, inserted into a eukaryotic expression vector, was stably transfected into Chinese hamster ovary (CHO) cells. Immunoprecipitation of cellular 35S-methionine-labeled proteins with Hashimoto's serum revealed a 105-101 kilodalton doublet. In contrast, cells transfected with wild-type hTPO yielded a 112-105 kilodalton doublet. In pulse-chase experiments, CHO cells expressing the truncated hTPO protein secreted immunoprecipitable TPO into the culture medium after 4 h of chase, with levels accumulating progressively over a 24-h period. In contrast, CHO cells expressing wild-type hTPO released no immunoprecipitable TPO into the culture medium. The secreted, truncated form of hTPO appeared as a single band of lesser electrophoretic mobility, as opposed to the doublet expressed within cells. TPO enzymatic activity was present in conditioned media from CHO cells transfected with the mutated hTPO, but was absent in media from cells expressing wild-type hTPO. The stability of the mutated protein appeared similar to that of wild-type hTPO. In summary, we have generated a mutated, secreted form of hTPO that is enzymatically active and immunologically intact. Our data confirm the existence of a transmembrane domain in hTPO, and that hTPO is predominantly an enzyme with an extracellular orientation. The secreted form of hTPO has the potential for generating large amounts of soluble TPO protein for use in future structural and immunological studies.