Characterization of four different mammalian-cell-derived recombinant human interferon-beta 1s. Identical polypeptides and non-identical carbohydrate moieties compared to natural ones

In order to evaluate the structural identification of various recombinant human interferon-beta 1s, the recombinant proteins were produced in four different mammalian cells (human PC12 and PC8 lung adenocarcinoma cells, Chinese hamster ovary cells and mouse C127 cells) and characterized. Each mammalian-cell-derived recombinant human interferon-beta 1 represented a single band of 23 kDa on sodium dodecyl sulphate/polyacrylamide gel electrophoresis, the same molecular mass as fibroblast-derived natural human interferon-beta 1. Specific activities, amino acid compositions, amino-terminal sequences, peptide maps on C18 reversed-phase high-performance liquid chromatography and circular dichroic spectra of recombinant proteins were in good agreement with natural ones. On the other hand, the patterns of isoelectric focusing were different between mammalian-cell-derived recombinant human interferon-beta 1s and natural human interferon-beta 1. Sugar composition analysis revealed that the recombinant protein from Chinese hamster ovary cells has a similar sugar composition to that of natural protein and the other recombinant proteins have increased amounts of galactose and glucosamine in comparison to the natural protein. Furthermore, there is no galactosamine in the natural protein, while small amounts of galactosamine were detected in the oligosaccharides released from PC8- and C127-derived recombinant proteins by N-glycanase. These results indicate that mammalian-cell-derived recombinant human interferon-beta 1s have identical polypeptides to those of natural human interferon-beta 1 but their carbohydrate moieties, including unusual N-linked oligosaccharides, are individually different from natural ones and depend on the host cell.     

Human recombinant lipocortin 1 inhibits prostacyclin production by human umbilical artery in vitro

Submicromolar concentrations of human recombinant Lipocortin 1 inhibit the release of prostacyclin from human umbilical artery rings in a dose-dependent fashion. This is the first demonstration that the recombinant protein is effective in human cells.     

Two monoclonal antibodies distinguish between human recombinant interferon-alpha 5s produced by Escherichia coli and by mouse cells

Two monoclonal antibodies against human IFN-alpha--one against natural leukocyte IFN-alpha and the other against recombinant human IFN-alpha 2 produced in E. coli--were prepared, and designated as HT-1, and 104-5-f, respectively. These monoclonal antibodies were used to examine the antigenicities of recombinant human IFN-alpha 5s produced by E. coli and by mouse cells. The HT-1 antibody could bind and neutralize recombinant human IFN-alpha 5 synthesized in mouse cells, but not recombinant human IFN-alpha 5 synthesized in E. coli. On the other hand, the 104-5-f antibody could bind and neutralize recombinant human IFN-alpha 5 synthesized in E. coli but not recombinant human IFN-alpha 5 synthesized in mouse cells. Then these monoclonal antibodies or sheep polyclonal antibody against human IFN-alpha were used to immunoprecipitate the radioactively labeled recombinant human IFN-alpha 5 synthesized either in E. coli or mouse cells, and analysed on polyacrylamide gel electrophoresis in the presence of NaDodSO4. The labeled recombinant human IFN-alpha 5 produced by mouse cells could be immunoprecipitated with the HT-1 monoclonal antibody or sheep anti-(human IFN-alpha) polyclonal antibody but not with the 104-5-f monoclonal antibody and showed a band of Mr. 17,500 on polyacrylamide gel electrophoresis in the presence of NaDodSO4. On the other hand, the labeled recombinant human IFN-alpha 5 produced by E. coli could be immunoprecipitated with the 104-5-f monoclonal antibody but not with the HT-1 monoclonal antibody and showed a band of similar Mr. on polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Furin-mediated processing of Pro-C-type natriuretic peptide

C-type natriuretic peptide (CNP) is a member of the natriuretic peptide family that is involved in a variety of homeostatic processes. Here we characterize the processing essential for the conversion of the precursor, human pro-CNP, to the biologically active hormone. In human embryonic kidney 293 and chondrosarcoma SW 1353 cells, recombinant pro-CNP was converted into a mature peptide intracellularly as detected by Western analysis. Expression of recombinant human corin, a proatrial natriuretic peptide convertase, did not enhance the processing of pro-CNP in these cells. The processing of pro-CNP was inhibited in the presence of an inhibitor of the endoprotease furin but was not affected by inhibitors of matrix metalloproteinases and tumor necrosis factor-alpha convertase. In furin-deficient human colon adenocarcinoma LoVo cells, no conversion of recombinant pro-CNP to CNP was detected. Expression of recombinant human furin in LoVo cells restored the ability of these cells to process pro-CNP. Furthermore, incubation of purified recombinant human furin with LoVo cell lysate containing pro-CNP led to the conversion of the precursor to a mature peptide. The furin-processed CNP was shown to be biologically active in a cell-based cGMP assay. These results demonstrate that furin is a critical enzyme for the processing of human pro-CNP.     

Distinct immunological and biochemical properties of thyroid peroxidase purified from human thyroid glands and recombinant protein produced in insect cells.

The biosynthesis of thyroid hormone from thyroglobulin is catalysed by thyroid peroxidase (TPO), an integral membrane protein. TPO is also a major autoantigen in autoimmune thyroid disease and autoantibodies to TPO are markers for disease activity. Large quantities of purified TPO are essential for elucidating its structure and understanding its role in disease activity. We describe the high yield purification of full-length recombinant human TPO from baculovirus infected insect cells and compare it to purified native TPO from human thyroid glands. In contrast to native human TPO, the human TPO produced in insect cells as a recombinant protein was insoluble and resistant to solubilisation in detergents. Reversible substitution of lysine residues with citraconic anhydride led to increased solubility of the recombinant TPO, allowing high-yield purification by monoclonal antibody chromatography. The purified enzyme preparation was shown to be TPO by its reactivity with monoclonal and polyclonal antibodies by enzyme linked immunosorbent assay and Western blotting. Both the human and recombinant purified TPO preparations also react with sera from patients with autoimmune thyroid disease, although the binding of conformational dependent autoantibodies was considerably lower to the recombinant TPO than to the native TPO. This suggests that the recombinant TPO may differ in some aspects of its tertiary structure. The purified recombinant TPO was devoid of enzyme activity, in contrast to the enzymatically active, purified human TPO preparations. Both preparations contained comparable amounts of haem (R(z)=0.269), but a shift in the Soret band of recombinant TPO (402 nm) from that of natural TPO (409 nm) indicates that the lack of enzymatic activity of the recombinant enzyme may be due to changes in the protein backbone surrounding the haem. Both the purified native and recombinant TPO, under non-denaturing conditions, show evidence of high molecular mass oligomers, although the latter preparation is prone to a greater degree of aggregation. In conclusion, our studies indicate that recombinant TPO generated in insect cells is conformationally distinct from the native TPO, is insoluble and enzymatically inactive, consistent with the difficulties associated with its purification and crystallisation.     

人视黄醇结合蛋白4在杆状病毒系统中的表达及其多克隆抗体制备

旨在利用杆状病毒系统表达、制备人视黄醇结合蛋白(RBP4)并检测其免疫原性。将人RBP4基因片段及信号肽SS64片段亚克隆到杆状病毒转移载体pFastBac-dual(pFBd)中,获得相应的重组转移质粒;转化大肠杆菌菌株DH10bac,转座后经筛选获得重组穿梭质粒rbacmid,将重组穿梭质粒转染孔板培养的Sf9细胞,获得含人RBP4表达框的重组杆状病毒,经过扩增获得毒种。毒种感染对数生长期的Sf9细胞并表达人RBP4蛋白(I-RBP4),通过SDS-PAGE和Western blotting对表达蛋白进行检测和鉴定。用毒种感染悬浮培养的Sf9细胞制备一批RBP4蛋白,完成SDS、Western blotting的检测及少量的多抗制备。纯化重组蛋白并与E.coli重组人RBP4(E-RBP4)分别免疫家兔。实验结果,酶切鉴定及测序证实重组转移质粒构建正确;成功构建重组RBP4-bacmid;人RBP4蛋白在昆虫细胞获得高效表达。表达的RBP4蛋白可以分泌到培养基中,分子量约为23 kDa,经过计算表达量为100 mg/L;纯化蛋白免疫兔子制备了多抗血清,血清滴度为1∶100 000,高于原核表达的抗体滴度(1∶10 000),与人体提纯蛋白制备的抗体滴度相近。杆状病毒系统高效表达了人的RBP4蛋白,具有较好的抗原性,并获得高亲和力的抗血清,为下一步的人血RBP4检测试剂盒的制备打下了坚实的基础。     

Recombinant glycodelin carrying the same type of glycan structures as contraceptive glycodelin-A can be produced in human kidney 293 cells but not in chinese hamster ovary cells.

We have produced human recombinant glycodelin in human kidney 293 cells and in Chinese hamster ovary (CHO) cells. Structural analyses by lectin immunoassays and fast atom bombardment mass spectrometry showed that recombinant human glycodelin produced in CHO cells contains only typical CHO-type glycans and is devoid of any of the N, N'-diacetyllactosediamine (lacdiNAc)-based chains previously identified in glycodelin-A (GdA). By contrast, human kidney 293 cells produced recombinant glycodelin with the same type of carbohydrate structures as GdA. The presence of a beta1-->4-N-acetylgalactosaminyltransferase functioning in the synthesis of lacdiNAc-based glycans in human kidney 293 cells is concluded to be the cause of the occurrence of lacdiNAc-based glycans on glycodelin produced in these cells. Furthermore, human kidney 293 cells were found to be particularly suited for the production of recombinant glycodelin when they were cultured in high glucose media. Lowering the glucose concentration and the addition of glucosamine resulted in higher relative amounts of oligomannosidic-type glycans and complex glycans with truncated antennae. Human glycodelin is an attractive candidate for the development of a contraceptive agent, and this study gives valuable information for selecting the proper expression system and cell culture conditions for the production of a correctly glycosylated recombinant form.     

Expression, characterization, and biochemical properties of recombinant human salivary amylase

Human salivary amylase, a major component of human salivary secretions, possesses multiple functions in the oral cavity. It is the only enzyme in saliva capable of degrading oligosaccharides, which are used by the oral microflora for nutritional purposes. In order to understand its role in disease processes such as caries, we have undertaken the structure-function analyses of amylase. In this regard, the nonglycosylated human salivary amylase was expressed in a baculovirus expression system. The native and the recombinant amylases exhibit similar biochemical as well as biophysical properties. Unlike recombinant human pancreatic amylase, recombinant human salivary amylase is not glycosylated when expressed in a baculovirus system as determined from the crystal structure determination of the recombinant enzyme. Therefore, this system is suitable for further structure-function work without resorting to enzymatic removal of the carbohydrate chain. Details of the expression, purification, and biophysical properties will be presented.     

人Artemin cDNA扩增及表达

以人胎脑RNA为模板, 采用RT-PCR技术扩增人Artemin cDNA。序列分析表明, 扩增的人Artemin cDNA核苷酸序列与已发表序列(GenBank登录号：AF115765)同源性为99.7%, 氨基酸序列同源性为100%。将经过序列分析确定的Artemin cDNA插入原核表达载体pGEX-6p-1中, 构建重组表达载体pGEX-6p-1-hART。通过SDS-PAGE分析重组人Artemin融合蛋白在大肠杆菌中的表达情况。结果表明, 重组人Artemin融合蛋白表达量约占宿主菌总蛋白的18.32%, 主要以包涵体形式存在。对表达的重组人Artemin融合蛋白包涵体进行溶解和复性, 并进行Western blotting分析。说明体外成功扩增人Artemin cDNA, 并在原核表达系统中高效表达了重组人Artemin融合蛋白。     

地高辛标记探针检测重组人干扰素β_(1b)中DNA残留量

为检测注射用重组人干扰素β1b半成品中外源性DNA残留量,以重组人干扰素β1b工程菌基因组DNA为模板,用地高辛标记探针,并以此探针进行点杂交。结果证明,该方法检测灵敏度较好,特异性较强,操作较安全简便,可用于重组人干扰素β1b制备过程中的质量监控及半成品的检定。     

Comparative analysis of the effects of the unpurified preparations of murine erythropoietin and human recombinant erythropoietin on erythroid and granulocyte-macrophage progenitor cells in semi-solid mouse bone marrow cultures]

Effects of unpurified murine erythropoietin and unpurified human recombinant erythropoietin on the growth of erythroid--BFU-E and granulocyte--macrophage progenitor cells--CFU--GM from the mouse bone marrow were compared using a methylcellulose culture system. Average erythropoietin titers for murine serum and supernatant human recombinant erythropoietin were 16 U/ml and 33 U/ml, respectively. The maximal stimulation was observed at 1-2 U/ml culture recombinant erythropoietin and 0.5 U/ml culture murine erythropoietin. Murine erythropoietin was more effective then human one. Murine and human recombinant erythropoietin had no significant effect on the number of CFU-GM colonies. But human recombinant erythropoietin could be preferentially used when studying the mechanism of erythropoiesis in man and animals because there were erythropoiesis inhibitors in mouse serum.     

Folding and activation of recombinant human prorenin

In vitro folding of mature renin, prorenin, and fused prorenin, all produced in denatured form in inclusion bodies in recombinant Escherichia coli, has been studied in order to evaluate the importance of prosequence in the folding of human renin. These studies have been compared with the in vivo folding and subsequent in vitro activation of recombinant human prorenin secreted by a nonbacterial expression system, namely Chinese hamster ovary (CHO) cells grown in serum-free medium. It is concluded that prosequence is essential in the folding of human renin and, therefore, the DNA coding for this sequence cannot be removed without affecting the recovery of active human renin from recombinant bacterial and nonbacterial systems.     

Single-step purification and structural characterization of human interleukin-6 produced in Escherichia coli from a T7 RNA polymerase expression vector

Human interleukin-6 or B-cell stimulatory factor-2 is a cytokine involved in acute phase and immune response. Cloning of cDNA for human interleukin-6 in the pT7.7 expression plasmid under the control of a bacteriophage T7 RNA polymerase promoter system allows rapid production of the cytokine in Escherichia coli. Upon cell induction with isopropyl thiogalactopyranoside, recombinant human interleukin-6 is overexpressed and forms insoluble inclusion bodies. Solubilization of the protein with 6 M guanidine hydrochloride and refolding in the presence of a reduction/oxidation system results in a quantitative recovery of recombinant human interleukin-6. This material is already 70% pure and can be further purified to homogeneity with a single passage over a weak anionic-exchange column. Extended structural characterization of the purified protein by electrospray mass spectrometry, automated Edman degradation and peptide mapping by high-pressure liquid chromatography/fast-atom-bombardment mass spectrometry demonstrates that recombinant human interleukin-6 is identical to the natural protein both in amino acid sequence and S-S bridge content. However, it contains a minor component accounting for about 20% of the entire translated protein which exhibits a Met-Ala dipeptide extension at the N-terminus. Purified recombinant human interleukin-6 is biologically active because it is able to induce at least 70-fold the human C-reactive promoter transfected in human hepatoma Hep 3B cells and is stable for several months in 10% glycerol at 4 degrees C. The expression system described in the present work has the main advantage of producing a high yield of recombinant human interleukin-6 (about 25 mg/l) combined with a very simple purification scheme.     

Retroviral mediated transfer and expression of human alpha 1-antitrypsin in cultured cells

Genetic deficiency of alpha 1-antitrypsin in man is a predisposing factor to emphysema and a disorder potentially correctable by somatic gene therapy. A full-length human alpha 1-antitrypsin cDNA was cloned into a retroviral vector and introduced into cells which package the recombinant gene in a retroviral capsule. Cells infected with the recombinant retrovirus express human alpha 1-antitrypsin mRNA and protein. The recombinant protein is glycosylated, secreted and exhibits anti-protease activity against human neutrophil elastase.     

Human recombinant laminin-binding protein: isolation, purification, and crystallization

The mRNA of the precursor of laminin-binding protein (LBP) was isolated from a human embryo kidney cell line and cloned. The determined sequence of the LBP gene showed complete identity with the LBP genes isolated from human lung and large intestine cells. The human LBP was expressed by E. coli cells, and it was purified using Ni-NTA-Sepharose chromatography. The mobility of the homogeneous recombinant human laminin-binding protein on SDS-PAGE was 43 kD. A mixture of eight murine monoclonal antibodies, the MPLR Pool against LBP, reacted with the recombinant LBP in Western blot. The interaction of the antiidiotypical antibodies 10H10 and E6B provided evidence that the epitope binding to protein E of the tick-borne encephalitis (TBE) virus is also preserved on the human recombinant LBP. Enzyme immunoassay confirmed the ability of the recombinant LBP to interact with protein E of TBE virus. The biological activity of the recombinant LBP allowed us to perform X-ray analysis of the spatial arrangement of the LBP molecule using the recombinant protein. For this purpose, crystals of the human LBP were obtained by the standing drop version of the pore diffusion technique. The crystals appropriate for X-ray structural analysis were 0.3 x 0.1 x 0.05 mm in size. The X-ray diffraction field of the crystal extended to 2.5 A.     

凝血因子Ⅶ及其重组表达新进展

凝血因子Ⅶ是一种维生素K依赖型的单链糖蛋白,在凝血过程中发挥着极其重要的作用,在临床上有广泛的应用,可用于伴有抑制物的血友病、先天性FⅦ缺乏症、血小板无力症及外科手术或严重外伤导致的创伤出血等止血用途.基因重组技术提供了能够大规模制备人凝血因子Ⅶ的有效途径,近年来已尝试并建立了多种人凝血因子Ⅶ的重组表达系统.对重组人凝...     

Preparation of recombinant alpha-thrombin: high-level expression of recombinant human prethrombin-2 and its activation by recombinant ecarin

We have established a large-scale manufacturing system to produce recombinant human alpha-thrombin. In this system, a high yield of alpha-thrombin is prepared from prethrombin-2 activated by recombinant ecarin. We produced human prethrombin-2 using mouse myeloma cells and an expression plasmid carrying the chicken beta-actin promoter and mutant dihydrofolate reductase gene for gene amplification. To increase prethrombin-2 expression further, we performed fed-batch cultivation with the addition of vegetable peptone in 50 liters of suspension culture. After five feedings of vegetable peptone, the expression level of the recombinant prethrombin-2 reached 200 micro g/ml. Subsequently, the recombinant prethrombin-2 could be activated to alpha-thrombin by recombinant ecarin expressed in a similar manner. Finally, recombinant alpha-thrombin was purified to homogeneity by affinity chromatography using a benzamidine-Sepharose gel. The yield from prethrombin-2 in culture medium was approximately 70%. The activity of the purified recombinant alpha-thrombin, including hydrolysis of a chromogenic substrate, release of fibrinopeptide A, and activation of protein C, was indistinguishable from that of plasma-derived alpha-thrombin. Our system is suitable for the large-scale production of recombinant alpha-thrombin, which can be used in place of clinically available alpha-thrombin derived from human or bovine plasma.     

Induction of alpha 1-acid glycoprotein by recombinant human interleukin-1 in rat hepatoma cells

The induction of alpha 1-acid glycoprotein mRNA by recombinant murine interleukin-1, recombinant human interleukin-1 alpha, and recombinant human interleukin-1 beta has been studied in the rat hepatoma cell line Fao. Whereas the stimulatory capacities of recombinant human interleukin-1 alpha and recombinant murine interleukin-1 were almost identical, the concentrations of recombinant human interleukin-1 beta needed for half-maximal induction of alpha 1-acid glycoprotein mRNA were lower by three orders of magnitude. A 60-fold increase in alpha 1-acid glycoprotein mRNA levels was observed 18 h after the addition of recombinant interleukin-1 beta. In parallel albumin mRNA levels decreased to about 30%. The alpha 1-acid glycoprotein mRNA induction was strictly dependent on the presence of dexamethasone. For a full stimulation dexamethasone concentrations of greater than 10(-7) M were needed, whereas concentrations of less than 10(-12) M were ineffective. The increase in alpha 1-acid glycoprotein mRNA after recombinant human interleukin-1 beta was followed by a 36-fold stimulation in alpha 1-acid glycoprotein synthesis and secretion. When protein synthesis was blocked by either cycloheximide, puromycin, or emetine, the induction of alpha 1-acid glycoprotein mRNA by recombinant human interleukin-1 beta was impaired suggesting the involvement of a short-lived protein in the induction of alpha 1-acid glycoprotein mRNA.     

Isolation of the human glyceraldehyde-3-phosphate dehydrogenase gene by hybridization with synthetic oligonucleotides

By screening a human genomic library from human lymphocyte DNA cloned in EMBL 3 vector with synthetic oligonucleotides homologous to the human GAPD cDNA 3'-non-coding region as probes, a unique lambda recombinant clone (EMBL-G5) was isolated at the Tm value. Preliminary restriction analysis and sequence data proved that this recombinant clone is the human structural gene for the glyceraldehyde- 3-phosphate dehydrogenase.     

Normal human epidermis contains an interferon-like protein

Interferons have been postulated to participate in growth regulation of normal body tissues and are known to inhibit growth of human epidermal keratinocytes in vitro. Polyclonal antibodies to recombinant human interferon-alpha, purified by passage over an affinity column (Sepharose coupled to the recombinant interferon), used in the indirect immunofluorescent method specifically stained the proliferative (basal) compartment of human epidermis in histological cross-sections of normal skin and in cultured keratinocyte colonies. Extracts prepared from healthy nonvirally infected keratinocyte cultures contained interferon activity as determined by viral plaque inhibition assay. Using the Western blotting technique column-purified antibodies and antisera to recombinant human interferon-alpha recognized a band of approximately 40 kD when reacted with both extracted keratinocyte proteins and recombinant human interferon-alpha standards, that gave in addition a band of approximately 20 kD. The above findings suggest that interferon or a closely related protein is present in the proliferative compartment of normal epidermis in the absence of viral infection and therefore may serve as a physiological modulator of epidermal growth.