Molecular cloning and nucleotide sequence of cDNA coding for calf preprochymosin.

DNA complementary to calf stomach mRNA has been synthesised and inserted into the Pst1 site of pAT153 by G-C tailing. Clones containing sequences coding for prochymosin were recognised by colony hybridisation with cDNA extended from a chemically synthesised oligodeoxynucleotide primer, the sequence of which was predicted from the published amino acid sequence of calf prochymosin. Two clones were identified which together contained a complete copy of prochymosin mRNA. The nucleotide sequence is in substantial agreement with the reported amino acid sequence of prochymosin and shows that this protein has a mol.wt. of 40431 and chymosin a mol.wt. of 35612. The sequence also indicates that prochymosin is synthesised as a precursor molecule, preprochymosin, having a 16 amino acid hydrophobic leader sequence analogous to that reported for other secreted proteins.     

Overexpression of binding protein and disruption of the PMR1 gene synergistically stimulate secretion of bovine prochymosin but not plant Thaumatin in yeast

When the heterologous proteins thaumatin and bovine prochymosin are produced in yeast cells as a fusion with the yeast invertase secretory signal peptide, less than 2% of the product is secreted in a biologically active form into the medium. The remainder accumulates intracellularly in a misfolded conformation. We investigated whether this poor secretion can be improved by overexpression of binding protein (BiP) one of the major chaperones in eukaryotic cells. Indeed, a tenfold increase in the level of binding protein, as a result of the introduction of extra copies of the kar2 gene into yeast cells containing a single, integrated copy of the invertase/prochymosin fusion gene, caused more than a 20-fold increase in the amount of extracellular prochymosin. By additional disruption of the PMR1 gene of these cells we were able to obtain secretion of virtually all of the prochymosin produced. Export of thaumatin, on the other hand, was not significantly stimulated by binding protein overexpression.     

Complete secretion of activable bovine prochymosin by genetically engineered L forms of Proteus mirabilis.

To circumvent problems encountered in the synthesis of active chymosin in a number of bacteria and fungi, a recombinant DNA L-form expression system that directed the complete secretion of fully activable prochymosin into the extracellular culture medium was developed. The expression plasmid constructions involved the in-frame fusion of prochymosin cDNA minus codons 1 to 4 to streptococcal pyrogenic exotoxin type A gene (speA') sequences, including the speA promoter, ribosomal binding site, and signal sequence and five codons of mature SpeA. Secretion of fusion prochymosin enzymatically and immunologically indistinguishable from bovine prochymosin was achieved after transformation of two stable protoplast type L-form strains derived from Proteus mirabilis. The secreted proenzyme was converted by autocatalytic processing to chymosin showing milk-clotting activity. In controlled laboratory fermentation processes, a maximum specific rate of activable prochymosin synthesis of 0.57 x 10(-3)/h was determined from the time courses of biomass dry weight and product formation. Yields as high as 40 +/- 10 micrograms/ml were obtained in the cell-free culture fluid of strain L99 carrying a naturally altered expression plasmid of increased segregational stability. The expression-secretion system described may be generally useful for production of recombinant mammalian proteins synthesized intracellularly as aberrantly folded insoluble aggregates.     

Complete secretion of activable bovine prochymosin by genetically engineered L forms of Proteus mirabilis

To circumvent problems encountered in the synthesis of active chymosin in a number of bacteria and fungi, a recombinant DNA L-form expression system that directed the complete secretion of fully activable prochymosin into the extracellular culture medium was developed. The expression plasmid constructions involved the in-frame fusion of prochymosin cDNA minus codons 1 to 4 to streptococcal pyrogenic exotoxin type A gene (speA') sequences, including the speA promoter, ribosomal binding site, and signal sequence and five codons of mature SpeA. Secretion of fusion prochymosin enzymatically and immunologically indistinguishable from bovine prochymosin was achieved after transformation of two stable protoplast type L-form strains derived from Proteus mirabilis. The secreted proenzyme was converted by autocatalytic processing to chymosin showing milk-clotting activity. In controlled laboratory fermentation processes, a maximum specific rate of activable prochymosin synthesis of 0.57 x 10(-3)/h was determined from the time courses of biomass dry weight and product formation. Yields as high as 40 +/- 10 micrograms/ml were obtained in the cell-free culture fluid of strain L99 carrying a naturally altered expression plasmid of increased segregational stability. The expression-secretion system described may be generally useful for production of recombinant mammalian proteins synthesized intracellularly as aberrantly folded insoluble aggregates.     

牛凝乳酶原基因的合成及其在乳酸克鲁维酵母中的表达

凝乳酶在奶酪加工中应用广泛,为获得高活性的凝乳酶制剂,采用乳酸克鲁维酵母为宿主,首次对经密码子优化的牛凝乳酶原基因进行表达。利用DNAWorks3.0软件辅助设计,用两步PCR法合成了小牛凝乳酶原基因(GenBank Accession No.AA30448)。将该基因插入酵母表达载体pKLAC1,构建了重组载体pKLAC1-Prochy,并用电脉冲法将线性化的重组质粒转化到乳酸克鲁维酵母GG799中。通过含1%酪蛋白的YEPD平板活性筛选,PCR鉴定,最后获得了一株多拷贝整合的基因工程菌chy1。该菌株可分泌表达牛凝乳酶原,经SDS-PAGE分析,证明重组牛凝乳酶原的分子量约为41kDa,符合预期大小,酸化处理后为36kDa,证明可以正确自我剪切。液体培养96h后,酶活最高达到99.67SU/mL。分别以半乳糖和葡萄糖为碳源的条件下表达,其酶活性差异不大,说明在发酵期间,可以不经过半乳糖诱导即可产生高水平的牛凝乳酶原产物。该工程菌的获得为进一步优化产酶条件及放大工艺提供了条件,并为凝乳酶的工业化生产奠定了基础。     

Examination of calf prochymosin accumulation in Escherichia coli: disulphide linkages are a structural component of prochymosin-containing inclusion bodies.

Recent reports have shown that synthesis of certain recombinant proteins in Escherichia coli results in the production of intracellular inclusion bodies. These studies have not analyzed the structure of the inclusion body especially regarding the intermolecular forces holding it together. We have examined structural aspects of inclusion bodies made in E. coli as a result of high level expression of the eukaryotic protein, calf prochymosin. Prochymosin is a monomeric protein containing three disulfide bridges. It was expressed at up to 20% of cell protein from a plasmid containing the E. coli tryptophan promoter, operator and ribosome binding site. Proteins in the inclusion bodies were analysed by Western blotting of SDS-polyacrylamide gels. When experiments were done using conditions which preserved the in vitro state of thiol groups, inclusions were shown to be composed of multimers of prochymosin molecules which were interlinked partly by disulfide bonds. The inclusion bodies also contained a high concentration of reduced prochymosin. The presence of intermolecular disulfides probably contributes to the difficulty of solubilizing recombinant prochymosin during its purification from E. coli.     

Enhancement of protein secretion in Pichia pastoris by overexpression of protein disulfide isomerase

A potential vaccine candidate, Necator americanus secretory protein (Na-ASP1), against hookworm infections, has been expressed in Pichia pastoris. Na-ASP1, a 45 kDa protein containing 20 cysteines, was directed outside the cell by fusing the protein to the preprosequence of the alpha-mating factor of Saccharomyces cerevisiae. Most of the protein produced by single copy clones was secreted outside the cell. However, increasing gene copy number of Na-ASP1 protein in P. pastoris saturated secretory capacity and therefore, decreased the amount of secreted protein in clones harboring multiple copies of Na-ASP1 gene. Overexpression of the endoplasmic reticulum (ER) resident, homologous chaperone protein, protein disulfide isomerase (PDI) was able to increase the secretion of (Na-ASP1) protein in high copy clones. The effect of PDI levels on secretion of Na-ASP1 protein was examined in clones with varying copy number of PDI gene. Increase in secreted Na-ASP1 secretion is correlated well with the PDI copy number. Increasing levels of PDI also increased overall Na-ASP1 protein production in all the clones. Nevertheless, there was still accumulation of intracellular Na-ASP1 protein in P. pastoris clones over-expressing Na-ASP1 and PDI proteins.     

Fermentation conditions for high-level expression of the tac-promoter-controlled calf prochymosin cDNA in Escherichia coli HB101

Escherichia coli HB101 harboring an expression plasmid that bears the calf prochymosin gene controlled by the tac promoter was cultivated under different conditions in order to find an optimal fermentation arrangement that would lead to maximal prochymosin yield. Our results indicate that it is advantageous to use lactose in the double role of inducer and carbon/energy source when foreign gene expression is controlled by the tac promoter and the gene product is only moderately toxic owing to its accumulation in the form of an intracellular body. Glucose, on the other hand, may be used when expression should be repressed. Growth temperature substantially influenced the specific rate of prochymosin and beta-lactamase gene expression and the plasmid copy number. Three phases were distinguished in the time course of the fermentation on lactose: exponential growth practically without prochymosin synthesis, linear growth with prochymosin synthesis, and prochymosin synthesis without growth of biomass. The synthesis of prochymosin in the form of intracellular inclusion body was accompanied by the loss of respiratory activity of the cell and the loss of its ability to multiply. Sixteen hours cultivation at 37 degrees C in a complex medium with lactose as inducer and carbon/energy source resulted in up to 30% of the volume and 48% of the total protein of biomass being accumulated for as prochymosin inclusion bodies. The concentration of extractable enzymatically active chymosin in the culture reached 12 mg/L.     

The effect of growth hormone on the proliferation of human Th cell clones

The effects of human growth hormone (hGH) on human Th cell clones were examined. Both 20K and 22K hGH stimulated the proliferation of Th2 and Th0 cells in the presence of mite antigen, whereas they did not stimulate the proliferation of Thl cells. Because the effect of 20K hGH was almost the same as that of 22KhGH, it was suggested that the action of hGH was not mediated through prolactin receptor but through hGH receptors. The application of growth hormone binding protein (GHBP) inhibited the cell growth of Th1 clones. In Th2 and Th0 cells GHBP inhibited the hGH-stimulated cell proliferation. However, GHBP alone did not affect the proliferation of Th2 and Th0 cells. hGH was detected in the supernatant of Th1 clones in the presence of mite antigen but it was not detected in Th2 clones. hGH was detected in one out of 4 batches of Th0 clones. These data indicated that hGH was secreted from Thl clones, and that Th0 clones possessed characteristics of both Th2 and Th0 clones.     

牛凝乳酶原基因在乳酸乳球菌中的表达

【目的】利用乳酸乳球菌nisin诱导基因表达系统(the NIsin Controlled gene Expression system,NICE)表达牛凝乳酶原。【方法】从克隆载体pS19-PPC中获得牛凝乳酶原基因,将该基因与表达载体pNZ8148连接并电转化乳酸乳球菌NZ9000,转化子经酶切、PCR和测序鉴定后,用nisin进行诱导表达,表达产物利用SDS-PAGE和Western blot鉴定,表达产物纯化后检测凝乳活性。【结果】重组牛凝乳酶原与天然牛凝乳酶原比较,其分子量大小、免疫性质、生物活性和抑制剂敏感性没有发现显著差异,其凝乳活性可达2×103IMCU/mL。【结论】在乳酸乳球菌中表达了具有凝乳活性的牛凝乳酶原,同时乳酸乳球菌作为发酵剂和凝乳酶产生菌双重角色的实现,为奶酪加工提供了新思路和新方法。     

Molecular cloning and the nucleotide sequence of cDNA for embryonic chicken pepsinogen: phylogenetic relationship with prochymosin

Embryonic chicken pepsinogen is an aspartyl proteinase that is specifically secreted during the embryonic period in the chicken proventriculus (glandular stomach). To learn the phylogeny of this pepsinogen, we isolated a cDNA clone by screening a lambda gt11 library of embryonic proventricular cDNAs with an antiserum to the embryonic chicken pepsinogen. We obtained a 200-base pair cDNA clone which encoded 18 amino acids that had high sequence homology with the carboxyl termini of other pepsinogens. Northern blot analysis revealed that this cDNA clone hybridized to a mRNA of 1,600 bases in the embryonic proventriculus but not to the mRNA in the adult proventriculus. The almost complete nucleotide sequence of embryonic chicken pepsinogen-cDNA was determined by sequencing longer cDNAs obtained by screening the same library with the 200-base pair cDNA and primer extension with a synthetic primer. The cDNA consisted of 1,281 nucleotides and encoded 383 amino acids for prepepsinogen. The predicted amino acid sequence was compared with the sequences of other aspartyl proteinases: pepsinogen A of human, monkey, pig, and chicken, progastricsin of monkey and rat, and bovine prochymosin. The phylogenetic tree constructed for them indicates the possibility that embryonic chicken pepsinogen diverged from prochymosin, after prochymosin and pepsinogen A had diverged from each other.     

Immunoglobulin production by a human-mouse somatic cell hybrid

The fusion of human lymphocytes and TEPC-15 mouse myeloma cells, which had not been adapted to culture, resulted in the establishment of in vitro hybrid cell cultures. Ten clones of this somatic cell hybrid were examined. There was preferential exclusion of human chromosomes: between two and five human chromosomes were identified in the hybrid clones by Giemsa banding. All of the clones had the mouse parental histocompatibility antigens, but only four clones also retained the human parental histocompatibility antigens. Secretion of parental immunoglobulin was determined by SDS-gel electrophoresis of species-specific immune precipitates. Synthesis of parental immunoglobulin by individual hybrid cells was determined by double label fluorescent antibody staining. Individual cells from six of the clones secreted and synthesized both human and mouse parental immunoglobulins. Three clones secreted only one parental immunoglobulin. Cells from one of these clones secreted and synthesized only human immunoglobulin. Cells from the remaining two clones secreted only one parental species of immunoglobulin but synthesized both human and mouse immunoglobulins. Finally, one clone did not secrete immunoglobulin, yet the individual cells synthesized both human and mouse parental species of immunoglobulin.     

人生长激素基因逆转录病毒载体的构建及其对3T3和ES细胞的转化与表达

为了研究生长激素基因在异源细胞中的表达,构建了携带人生长激素基因（ｈＧＨ）的逆转录病毒载体ｐＩＮＳ－ＧＨ,并将其导入小鼠成纤维细胞３Ｔ３和小鼠胚胎干细胞（ＥＳ细胞）ＣＣＥ中。ｐＩＮＳ－ＧＨ转化３Ｔ３细胞,获得了高效表达的克隆,即用放射免疫法检测到克隆培养基中有大量的人生长激素蛋白富积,如ＧＨＳＮＣ２０,富积率高达３７８４ｎｇ／ｍｌ,而且外源基因的整合很稳定。不同的３Ｔ３转化克隆的表达率有显著的差异,Ｓｏｕｔｈｅｒｎ杂交的结果表明,这种差异与外源基因整合的拷贝数无关。ｐＩＮＳ－ＧＨ转化ＣＣＥ细胞没有获得明显表达的克隆,而且外源基因的整合也不稳定。转化的ＥＳ细胞克隆总ＤＮＡ的Ｓｏｕｔｈｅｒｎ杂交结果表明,ｈＧＨ基因的确整合到细胞基因组中。目前尚未发现明显的外源ＤＮＡ重排的迹象。     

Sta!le and transient expression of mouse submaxillary gland renin cDNA in AtT20 cells: proteolytic processing and secretory pathways

Apart from kidney, where renin synthesis takes place in all mammals, the submaxillary gland (SMG) of most mouse strains constitutes an important source of an isoenzyme, renin-2, that is highly homologous to renal renin, but unglycosylated [(1982) Nature 298, 90-92]. This unique phenotype is due to the presence of an extra copy of th renin gene. A puzzling observation is that (pro)renin-2 cannot be detected in the kidney of these animals, although both mRNAs accumulate at similar levels [(1985) Proc. Natl. Acad. Sci. USA 82, 6196-6200]. In order to investigate whether (pro)renin-2 expression is detectable in mouse heterologous cell lines we transfected the renin-2 cDNA into AtT20 (pituitary corticotrope) and BTG9A (hepatoma) cells. Stable clones expressing renin were obtained in both cases. BTG9A cells secreted only prorenin while AtT20 cells secreted prorenin and active renin. In addition, in AtT20 cells the secretion of active renin was stimulated by 8-Br cAMP. Our results show that unglycosylated (pro)renin-2 can be expressed and secreted in two murine cell lines. Moreover, it is correctly processed to active renin and secreted upon stimulation in AtT20 cells.     

牛凝乳酶原基因克隆及序列的进化分析

利用RT-PCR克隆获得牛凝乳酶原基因的cDNA序列, 测序后与GenBank中凝乳酶原基因进行序列比对和生物信息学分析。序列比对统计分析显示, 该基因为牛凝乳酶原B基因, 与已知牛和其他哺乳动物的凝乳酶原基因具有很高的同源性,18种哺乳动物凝乳酶原基因密码子一、二、三位点的碱基偏倚度分别为：6.227、1.042和1.456。这表明该基因具有整体保守性和突变位点偏倚性两个特征, 可以作为哺乳动物系统进化的研究对象。采用多种方法构建的该基因系统进化树一致表明, 偶蹄动物与灵长动物的亲缘关系比偶蹄动物与啮齿动物的亲缘关系更近, 比偶蹄动物与食肉动物的亲缘关系更远, 并且18种哺乳动物的亲缘关系与动物种系进化关系一致, 为哺乳动物系统进化关系研究提供了分子水平的佐证和依据。     

Simultaneous production of IL-2, IL-4, and IFN-gamma by activated human CD4+ and CD8+ T cell clones

In the present study, we have investigated the ability of human T cells to secrete IL-2, IL-4, and IFN-gamma. IL-4 and IFN-gamma were quantified with enzymatic immunoassays and IL-2 with a biologic assay by using the murine IL-2-dependent cell line CTLL-2. PBL, stimulated with Con A or with a combination of the phorbol ester 13-O-tetradecanoylphorbol-12-acetate and the Ca2+ ionophore A23187 secreted IL-2, IL-4, and IFN-gamma. The kinetics of the secretion of the three lymphokines was investigated with two CD4+ clones; one (GEO-2) that produced IL-2, IL-4, and IFN-gamma and another (HY640), that produced only IL-2 and IFN-gamma. Significant IL-2, IL-4, and IFN-gamma production was observed after only 8 h of activation. Maximal levels of IL-2 and IL-4 were found 20 h after the onset of the stimulation which subsequently decreased. In contrast, IFN-gamma levels continued to increase in a period up to 40 h and then leveled off. In spite of these differences in secretion, the kinetics of accumulation of mRNA did not differ. The IL-2, IL-4, and IFN-gamma mRNA were detectable 2 h after stimulation and continued to accumulate for a period up to 20 h. In a series of 22 CD4+ clones, 21 were able to secrete all three lymphokines upon stimulation. Almost all CD8+ clones were able to produce IL-2 and IFN-gamma, but only six of the 23 CD8+ T cell clones secreted IL-4. In addition, five CD4+ (allo)antigen-specific T cell clones were tested for IL-2, IL-4, and IFN-gamma secretion upon specific stimulation. Two alloantigen-specific and two tetanus toxoid-specific T cell clones secreted IL-2, IL-4, and IFN-gamma simultaneously, whereas one alloantigen-specific T cell clone secreted IL-2 and IFN-gamma, but not IL-4. A supernatant of the CD4+ T cell clone GEO-2, that contained high levels of IFN-gamma and IL-4, was unable to induce the low affinity receptor for IgE, CD23, on a Burkitt lymphoma cell line. However, after separation of IL-4 from IFN-gamma by using HPLC, the IL-4-containing fraction-induced CD23, which could be blocked by the fraction that contained IFN-gamma and by a polyclonal rabbit anti-IL-4 antiserum. Finally, the partly purified IL-4, that was devoid of IL-2, promoted the growth of the clone GEO-2.     

Automatic laboratory-scale fed-batch procedure for production of recombinant proteins using inducible expression systems of Escherichia coli

Summary An automatic fed-batch procedure for the production of recombinant proteins in Escherichia coli was developed. Using glycerol as carbon source and by controlling the growth rate by using feed-forward algorithm, enabled high specific expression level (10–20 % of total cell protein) at high cell densities (20 g dry wt/l) to be achieved: rat and human soluble catechol-O-methyltransferase, calf prochymosin, and human troponin C were expressed with nearly 50-fold higher volumetric yield compared to the conventional (batch) procedures.     

Production of Chymosin in Escherichia coli Cells and Its Enzymatic Properties

The production of prochymosin directed by a cloned cDNA under the control of a trp promoter was examined in E. coli C600 and HB101. The latter host exhibited a higher degree of expression as to the production of prochymosin in the form of inclusion bodies, which accounted for more than 15 ~ 20% of the total cellular protein. The conditions for the processing of prochymosin in the inclusion bodies to active chymosin were determined. Several enzymatic properties of the processed bacterial chymosin, such as its specific activities as to milk-clotting and proteolysis, heat stability and Ca^{2 +} dependence of the clotting activity, were almost identical to those of authentic chymosin. However, a slight difference was observed with regard to the immunological reactivity with anti-prochymosin antibody.