Activation studies of the multiple forms of prochymosin (prorennin).

Activation of the four separate components of prochymosin (prorennin) at pH 5.0 demonstrated that each zymogen was the precursor to an electrophoretically distinct chymosin (rennin). When the increase in milk-clotting activity with time was analysed, the mechanism of activation of unfractionated prochymosin, individual prochymosin components, and a mixture of the prochymosin fractions at pH 5.0 was shown to follow essentially autocatalytic kinetics. The activation of prochymosin C was completed in 70 h, whereas the other three fractions each required more than 110 h for complete activation under the same conditions. Intact prochymosin, the mixture of four components and prochymosin C were activated at similar rates. Interaction of the individual fractions during activation is suggested to explain the increased rate of the activation for the mixture. Comparison of autocatalytic activation of unfractionated prochymosin purified chromatographically at pH 6.7 and 5.7 demonstrated an increased rate of reaction of the zymogen prepared at the lower pH value. The possibility that prochymosin became susceptible to activation during preparation at pH values slightly below 6.0, as a result of changes in the proportion of the components or a conformational change and exposure of the active site, is discussed.     

Fractionation and isolation of the multiple forms of prorennin (prochymosin)

The heterogeneity of prorennin was studied by chromatography on DEAE-cellulose and microgranular DEAE-cellulose columns, as well as by polyacrylamide-gel electrophoresis. Prorennin prepared by alum treatment, salting-out and chromatography was resolved into three components by a compound gradient of sodium phosphate on microgranular DEAE-cellulose. Polyacrylamide-gel electrophoresis confirmed the chromatographic results, but crystalline rennin was shown to consist of four bands. When prorennin was isolated directly by chromatography, four zymogen components were resolved on microgranular DEAE-cellulose with a modified compound gradient of sodium phosphate. Polyacrylamide-gel electrophoresis confirmed the existence of four multiple forms of prorennin as well as homogeneity of the chromatographic fractions.     

Investigations on the activation of bovine prochymosin.

Activation of prochymosin at pH below 2.5 results in formation of the active enzyme pseudochymosin by proteolytic cleavage of the bond 27--28. Pseudochymosin is 15 amino acid residues longer than chymosin. It is the final activation product at low pH, whereas chymosin is formed by activation between pH 4 and 5. Pseudochymosin is converted to chymosin when it is brought to pH 5.5. Our present knowledge does not allow quantitative evaluation of the possible reactions involved in formation of pseudochymosin, but the course of activation at pH 2 is in accordance with an intermolecular reaction between two zymogen molecules as the predominant reaction. We find indications of an intramolecular reaction when intermolecular reactions are prevented by immobilization of the zymogen.     

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。     

Crystallization and preliminary diffraction studies of recombinant human granulocyte-stimulating factor (KW2228).

Human granulocyte colony-stimulating factor (hG-CSF) specifically stimulates proliferation of neutrophils. Two crystal forms of a mutant of hG-CSF expressed in Escherichia coli have been obtained using the hanging drop vapour diffusion method. One form is triclinic, space group P1, with cell dimensions a = 37.3 A, b = 46.4 A, c = 47.7 A, alpha = 105.5 degrees, beta = 98.0 degrees and gamma = 109.4 degrees. The other is monoclinic, space group C2, with cell dimensions a = 82.0 A, b = 49.2 A, c = 49.4 A and beta = 113.9 degrees. Both crystal forms diffract beyond 2.0 A and are suitable for X-ray analysis.     

Biological equivalence of natural bovine and recombinant human alpha-endothelial cell growth factors

The cDNA encoding human alpha-endothelial cell growth factor (alpha-ECGF) has been engineered for high-level expression in Escherichia coli. Induction of bacterial cultures harboring the recombinant plasmid pMJ26 results in the appearance of a prominent 16-kDa polypeptide. This protein has been purified from bacterial lysates using a rapid, 2-step procedure employing heparin-Sepharose affinity based chromatography and reversed-phase high pressure liquid chromatography. Recombinant human alpha-ECGF was compared to bovine brain-derived alpha-ECGF in three biological assays: receptor binding on murine lung capillary endothelial cells (LE-II cells), stimulation of [3H]thymidine incorporation in LE-II cells, and stimulation of human umbilical vein endothelial cell proliferation. The results demonstrate that the recombinant human mitogen has the same biological potency as the bovine brain-derived material. Fluorescence spectroscopy was used to study the interaction between recombinant ECGF and heparin. Heparin-binding resulted in a 40% reduction in the intrinsic fluorescence of ECGF, consistent with a heparin-induced conformational change. The intrinsic fluorescence of ECGF also varied as a function of pH.     

Crystallization and preliminary X-ray diffraction studies of bovine recombinant immune interferon

Reproducible conditions have been established for the crystallization of recombinant bovine immune interferon. Two cystalline forms of this protein were obtained. A tetragonal form, space group P422, with unit cell dimensions a = b = 59.0 A and c = 125.7 A and an orthorhombic form, space group P2(1)2(1)2(1), with unit cell dimensions a = 42.80 A, b = 79.90 A and c = 85.64 A were obtained under similar crystallization conditions. The orthorhombic form diffracts to 2.6 A resolution, contains a single interferon dimer in the asymmetric unit of structure and is suitable for X-ray diffraction analysis.     

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。     

Denaturation of apolipoprotein A-I and the monomer form of apolipoprotein A-I(Milano).

In this study the thermal and denaturant induced unfolding of apolipoprotein A-I (apo A-I) and the monomer form of apolipoprotein A-I(Milano) (apo A-I(M)) was followed. Dimer apo A-I(M) was reduced with dithiothreitol, which was present in the protein solutions in all experiments. Thermal denaturation is followed by differential scanning calorimetry (DSC) and far-UV and near-UV CD. Both apo A-I and monomer apo A-IM have a broad asymmetric DSC peak that could be deconvoluted into three non two-state transitions, apo A-I being more stable than the monomer apo A-IM. Estimation of melting of tertiary structure by near-UV CD is lower than that for secondary structure determined from far-UV. This together with the non two-state unfolding of the proteins observed with DSC is indicative of unfolding via a molten globular-like state. Apo A-I and monomer apo A-I(M) are equally susceptible to guanidinum chloride, half-unfolded at 1.2 M denaturant. The presence of 0.5 and 1.0 M denaturant, lower and equalize the denaturation temperatures of the proteins, respectively.     

Bioactive recombinant methionyl bovine prolactin: structure-function studies using site-specific mutagenesis

A method has been developed for the extraction from transformed Escherichia coli cells of methionyl bovine PRL (met-bPRL) in a relatively pure form. While the extracted met-bPRL was as reactive as the native hormone with respect to polyclonal anti-bPRL antibodies, its bioactivity, as measured by the Nb2 lactogen in vitro bioassay, was relatively low. The bioactivity of the met-bPRL could be increased to the same order as that of the native hormone by treatment with a mixture of oxidized and reduced thioredoxin. A number of variant met-bPRLs containing specific amino acid changes have been generated by site-specific mutagenesis. The changes involved the substitution (or deletion) of some of the conserved amino acids in bPRL by the different amino acids present at the corresponding positions in the related, but nonlactogenic bovine GH. Nine mutants containing single amino acid changes had bio- and immunoactivities of the same order as those of met-bPRL. One mutant, which incorporated two of the single amino acid changes (serine 62 to threonine and threonine 65 to alanine), had immunoactivity approximating that of met-bPRL but much lower bioactivity (45%). A further mutant, generated by the deletion of tyrosine 28, had essentially no bioactivity although it could not be distinguished immunologically from met-bPRL or bPRL. The findings are discussed in the light of the putative three-dimensional PRL structure and current hypotheses which seek to relate specific regions of PRL to lactogenic activity. It appears that the first putative alpha-helix of bPRL is important for the binding and mitogenic activity of the hormone.     

Functional implications of disulfide bond, Cys206-Cys210, in recombinant prochymosin (chymosin)

Prochymosin (chymosin) contains three disulfide bonds: Cys45-Cys50, Cys206-Cys210, and Cys250-Cys283. We have demonstrated that Cys250-Cys283 is indispensable for correct refolding of prochymosin, whereas Cys45-Cys50 is dispensable but has some contribution to the stability and substrate specificity of the enzyme. Here, we report the results about the functions of Cys206-Cys210 by site-directed mutagenesis studies. In a glutathione redox system C206A/C210A mutant exhibited oxidative refolding kinetics and efficiency ( approximately 40% reactivation) similar to those of the wild-type prochymosin, indicating that Cys206-Cys210 is also dispensable for refolding. However, C206S/C210S and single-site mutants (C210A, C210S, and C206A) showed only about 3 and 0-0.4% reactivation, respectively. This is quite different from the Cys45-Cys50 deficient mutants (C45A, C50A, C45A/C50A, C45D, C50S, C45D/C50S, C45A/C50S), which have comparable refolding efficiencies, implying that the substituents at position 206 and 210 play more important role in determining correct refolding than those at position 45 and 50. Urea-induced denaturation and fluorescence quenching studies indicated that the prochymosin mutants C206A/C210A and C206S/C210S were 2.1 and 4.8 kJ/mol less stable than prochymosin and some tryptophan residue in the mutated molecules was less exposed. However, the wild-type and mutant prochymosins shared similar far-UV CD and fluorescence emission spectra and similar specific potential activity, suggesting that the overall conformation was maintained after mutation. Activity assay and kinetic analysis revealed that mutation did not change the specific milk-clotting activity significantly but resulted in an increase in K(m) and k(cat) toward a hexapeptide substrate. On the basis of the above-mentioned perturbance of tryptophanyl microenvironment and the three-dimensional structure of chymosin, we proposed that deletion of Cys206-Cys210 may induce a propagated conformational change, resulting in a perturbance of the local conformation around active-site cleft and in turn, an alteration of the substrate specificity.     

大肠杆菌异源重组蛋白质的变性与复性

描述了大肠杆菌异源重组蛋白质的形成、制备、变性和复性,综述了国内外变性、复性的有效方法。     

Androgens in the bovine fetus and dam (38567).

Umbilical arterial and venous blood, and fetal testes were taken from 38 bovine fetuses at 90, 180 or 260 days of gestation. Concurrently blood also was taken from the jugular, and from the uterine artery and vein of the dams. Testosterone and androstenedione were determined by radioimmunoassays. Fetal testicular homogenates had 0.96 and 0.35 mug/g of testosterone and 0.39 and 0.50 mug/g of androstenedione at 180 and 260 days of gestation, respectively. Males had five to tenfold more serum testosterone and about twofold more androstenedione than female fetuses at each trimester of gestation. Male fetal blood testosterone decreased (P less than 0.01) from 2.7 to 0.3 ng/ml between 90 and 260 days of gestation. But, maternal testosterone and androstenedione increased (P less than 0.05) during gestation in cows with males, but not in cows with female fetuses. Testosterone was higher (P less 0.05) in cows carrying males than in cows with female fetuses. Androstenedione was higher in blood leaving the placenta on both the maternal and on the vetal sides suggesting placental synthesis of androstenedione.     

Native and recombinant bovine placental lactogens

The bovine placenta produces a wide variety of proteins that are structurally and functionally similar to the pituitary proteins from the GH/PRL gene family. Bovine placental lactogen (bPL) is a 200-amino acid long glycoprotein hormone that exhibits both lactogenic and somatogenic properties. The apparent molecular masses of purified native (n) bPL molecules (31-33 kDa) exceed 23 041 Da, which is the theoretical molecular mass of the protein core. At least six isoelectric variants (pI: 4.85-6.3) of bPL were described in cotyledonary extracts and three different bPL isoforms (pI: 4.85-5.25) were found in fetal sera. The bPL molecules that are detected in higher concentrations in peripheral circulation exhibit a more acidic pI than those present in placental homogenates. This may reflect an important glycosylation process occurring just prior to the bPL secretion. The bPL mRNA is transcribed in trophectoderm binucleate cells starting from Day 30 of pregnancy until the end of gestation. In mothers, bPL is involved in the regulation of ovarian function, mammogenesis, lactogenesis, and pregnancy stage-dependent adaptation of nutrient supplies to the fetus. Due to the higher fetal, compared to maternal concentrations of circulating hormone, it has been suggested that bPL primarily targets fetal tissues.