Expression in insect cells and purification of a catalytically active recombinant human gastric lipase

Human gastric lipase (HGL) cDNA was synthesized by RT-PCR amplificationand cloned into the PVL 1392 baculovirus transfer vector. Therecombinant transfer vector was cotransfected with a modifiedbaculovirus DNA (Baculogold^TM) which contains a lethal deletion.Cotransfection of baculovirus DNA with the recombinant transfervector rescues the lethal deletion of this virus DNA and reconstitutesviable virus particles inside the transfected insect cells.BTI-TN-5B1-4 insect cells (also called High Five^TM cells) wereused to express recombinant HGL. The level of HGL secretionwas {small tilde}32 mg/1 of culture medium. The insect cellsalso accumulated HGL intracellularly, which indicated the existenceof rate-limiting steps in the secretion of HGL. Therefore weinvestigated the effect of replacing the HGL signal peptide(SP) by other SP of secreted proteins. The honeybee melittinSP and the human pancreatic lipase (HPL) SP were tested. Thefusion of HGL with HPL SP resulted in a 2-fold increase in theamount of lipase secreted from the insect cells. The recombinantactive HGL was not processed at the expected cleavage site ofthe natural enzyme, however, but at residue +3. On the otherhand, High Five^TM cells transfected with the vector encodingHGL fused to the melittin SP did not secrete any detectableactive HGL. Recombinant HGL was identified using the Westernblot procedure with rabbit polyclonal antibodies. The proteinmigrated with an apparent molecular mass of 45 kDa under SDS–PAGEanalysis (compared with 50 kDa in the case of natural HGL),indicating that the insect cells have only a limited capacityto glycosylate HGL. The maximum specific activities of the recombinantlipase were 434, 730 and 562 units/mg using long-chain (Intralipid^TM),medium-chain (trioctanoylglycerol) and short-chain (tributyroylglycerol)triacylglycerols, respectively.     

负载型KI催化甘油与CO2合成甘油碳酸酯

添加不同组分对氧化铝载体进行调变改性,再以改性氧化铝为载体,负载KI制备了一系列负载型催化剂KI/Al₂O₃-MgO、KI/Al₂O₃-ZnO、KI/Al₂O₃-TiO₂和KI/Al₂O₃-ZrO₂,并通过CO₂、环氧丙烷和甘油合成甘油碳酸酯反应评价其催化活性,发现KI/Al₂O₃-MgO具有最高的活性。由不同载体的CO₂-TPD分析可以发现,载体表面少量碱性位的存在有利于反应进行。实验研究了不同负载量KI/Al₂O₃-MgO的活性及稳定性,发现KI负载量为1.5mmol/g较为适宜。同时,实验又通过N₂吸附/脱附(BET)、X射线衍射(XRD)等手段对不同负载量的KI/Al₂O₃-MgO进行了表征,进一步说明了负载量过多会导致KI晶粒团聚,并阻塞载体孔道。优化了反应条件,在最佳条件下(环氧丙烷0.3mol,甘油0.1mol,反应温度130℃,反应时间2h,反应压力6.0MPa),甘油的转化率为65.5%,甘油碳酸酯的产率为60.8%。     

Construction and structure-activity relationships of chimeric prourokinase derivatives with intrinsic thrombin-inhibitory potential

The blood clotting enzyme thrombin plays a central role in theaetiology of occlusive disorders such as stroke and acute myocardialinfarction. During fibrinolytic therapy with plasminogen activators,thrombin is neutralized by anticoagulative drugs. In order tocombine plasminogen-activating and thrombin-inhibitory activitieswe constructed chimeric derivatives of recombinant single-chain,urokinase-type plasminogen activator (rscu-PA) which comprisethe kringle and protease domain of rscu-PA fused via a linkersequence to a thrombin-inhibitory domain. The inhibitory domaincontains a sequence element directed to the active site of thrombinand a sequence taken from either hirudin or the human thrombinreceptor both binding to the fibrinogen recognition site ofthrombin. Analysing different sets of point mutants showed thatthe linker between the protease domain and the active sitedirectedsequence is contributing significantly to the thrombin-inhibitorypotential. Kinetic analysis of thrombin inhibition revealedthat most of the chimeras tested competitively inhibit the thrombin-mediatedcleavage of a peptide substrate in a concentration-dependentmanner; however, in two examples the insertion of one glycineresidue into the active site directed-sequence abolished theblockade of the active site. This supports the conclusion thatthe chimeras with high thrombin-inhibitory potential interactwith the active site and the fibrinogen recognition site ofthrombin.     

The structure of E.coli soluble inorganic pyrophosphatase at 2.7 A resolution

Hie structure of E.coli soluble inorganic pyrophosphatase hasbeen refined at 2.7 resolution to an R-factor of 20.9. Theoverall fold of the molecule is essentially the same as yeastpyrophosphatase, except that yeast pyrophosphatase is longerat both the N- and C-termini. Escherichia coli pyrophosphataseis a mixed +ß protein with a complicated topology.The active site cavity, which is also very similar to the yeastenzyme, is formed by seven ß-strands and an -helixand has a rather asymmetric distribution of charged residues.Our structure-based alignment extends and improves upon earliersequence alignment studies; it shows that probably no more than14, not 15–17 charged and polar residues are part of theconserved enzyme mechanism of pyrophosphatases. Six of theseconserved residues, at the bottom of the active site cavity,form a tight group centred on Asp70 and probably bind the twoessential Mg⁺ ions. The others, more spreadout and more positivelycharged, presumably bind substrate. Escherichia coli pyrophosphatasehas an extra aspartate residue in the active site cavity, whichmay explain why the two enzymes bind divalent cation differently.Based on the structure, we have identified a sequence motifthat seems to occur only in soluble inorganic pyrophosphatases.     

Highly enantioselective kinetic resolution of two tertiary alcohols using mutants of an esterase from Bacillus subtilis

Enzyme-catalyzed kinetic resolutions of secondary alcohols are a standard procedure today and several lipases and esterases have been described to show high activity and enantioselectivity. In contrast, tertiary alcohols and their esters are accepted only by a few biocatalysts. Only lipases and esterases with a conserved GGG(A)X-motif are active, but show low activity combined with low enantioselectivity in the hydrolysis of tertiary alcohol esters. We show in this work that the problematic autohydrolysis of certain compounds can be overcome by medium and substrate engineering. Thus, 3-phenylbut-1-yn-3-yl acetate was hydrolyzed by the esterase from Bacillus subtilis (BS2, mutant Gly105Ala) with an enantioselectivity of E = 56 in the presence of 20% (v/v) DMSO compared to E = 28 without a cosolvent. Molecular modeling was used to study the interactions between BS2 and tertiary alcohol esters in their transition state in the active site of the enzyme. Guided by molecular modeling, enzyme variants with highly increased enantioselectivity were created. For example, a Glu188Asp mutant converted the trifluoromethyl analog of 3-phenylbut-1-yn-3-yl acetate with an excellent enantioselectivity (E > 100) yielding the (S)-alcohol with > 99%ee. In summary, protein engineering combined with medium and substrate engineering afforded tertiary alcohols of very high enantiomeric purity.     

The crystal structure of {alpha}-bungarotoxin at 2.5 ? resolution: relation to solution structure and binding to acetylcholine receptor

We report collection of 2.5 ? resolution X-ray diffraction datafrom newly grown crystals of the rare ‘small unit cell’form of the long snake neurotoxin, -bungarotoxin. The previousmodel of the molecule has been rebuilt, and refined using least-squaremethods to a crystallographic residual of 0.24 at 2.5 ? resolution.-Bungarotoxin's crystal structure is compared with the crystalstructures of two other snake neurotoxins (cobratoxin and erabutoxin),and with its solution structure inferred from spectroscopicstudies. Significant differences include less ß-sheetin bungarotoxm's crystal structure than in solution, or in thecrystal structures of other neurotoxins, and an unusual orientationin the crystal of the invariant tryptophan. The functional,binding surface of bungarotoxin is described; it consists primarilyof hydrophobic and hydrogen-bonding groups and only a few chargedside chains. The structure is compared with experimental bindingparameters for neurotoxins.     

KF/C催化甘油酯交换合成碳酸甘油酯

以三嵌段共聚物(EO-PO-EO)F127为结构导向剂,甲阶酚醛树脂为碳源,KF·2H₂O为无机前驱体,用溶剂诱导挥发自组装的方法合成KF/C复合材料。采用XRD、BET和XPS等手段对合成的材料进行结构表征,并考察KF/C在甘油与碳酸二甲酯酯交换合成碳酸甘油酯反应中的性能,结果表明,在甘油加入量为0.184 6 g、碳酸二甲酯加入量为0.900 7 g、溶剂N,N-二甲基乙酰胺为5.426 g、催化剂KF/C加入量为0.1 g、反应温度100 ℃、反应时间2 h和搅拌速率600 r·min^-1条件下,甘油转化率达98.5%,碳酸甘油酯选择性达99.8%,催化剂具有较好的循环使用性能。     

The refined crystal structure of subtilisin Carlsberg at 2.5 ? resolution

We report here the X-ray crystal structure of native subtilisinCarlsberg, solved at 2.5 ? resolution by molecular replacementand refined by restrained least squares to a crystal-lographicresidual of 0.206. we compare this structure to the crystal structure of subtilisin BPN'. We find that, despite82 amino acid substitutions and one deletion in subtilisin Carlsbergrelative to subtilisin BPN', the structures of these enzymesare remarkably similar. We calculate an r.m.s. difference betweenequivalent a-carbon positions in subtilisin Carlsberg and subtilisinBPN' of only 0.55 ?. This confirms previous reports of extensivestructural bomology between these two subtilisins based on X-raycrystal structures of the complex of eglin-c with subtilisinCarlsberg [McPhalen,C.A., Schnebli.H.P. and James,M.N.G. (1985)FEBS Lett., 188, 55; Bode,W., Papamokos,E. and Musil,D. (1987)Eur. J. Biochem., 166, 673-692]. In addition, we find that thenative active sites of subtilisins Carlsberg and BPN' are virtuallyidentical. While conservative substitutions at residues 217and 156 may have subtle effects on the environments of substrate-bindingsites SI' and SI respectively, we find no obvious structuralcorrelate for reports that subtilisins Carlsberg and BPN' differin their recognition of model substrates. In particular, wefind no evidence that the hydrophobic binding pocket SI in subtilisinCarlsberg is ‘deeper’, ‘narrower’ or'less polar' than the corresponding binding site hi subtilisinBPN' [Karasaki and Ohno (1978) J. Biochem., Tokyo, 84, 531–538].     

Crystal structure of human serum albumin at 2.5 A resolution

A new triclinic crystal form of human serum albumin (HSA), derivedeither from pool plasma (pHSA) or from a Pichia pastoris expressionsystem (rHSA), was obtained from polyethylene glycol 4000 solution.Three-dimensional structures of pHSA and rHSA were determinedat 2.5 Å resolution from the new triclinic crystal formby molecular replacement, using atomic coordinates derived froma multiple isomorphous replacement work with a known tetragonalcrystal form. The structures of pHSA and rHSA are virtuallyidentical, with an r.m.s. deviation of 0.24 Å for allC atoms. The two HSA molecules involved in the asymmetric unitare related by a strict local twofold symmetry such that theC atoms of the two molecules can be superimposed with an r.m.s.deviation of 0.28 Å in pHSA. Cys34 is the only cysteinewith a free sulfhydryl group which does not participate in adisulfide linkage with any external ligand. Domains II and IIIboth have a pocket formed mostly of hydrophobic and positivelycharged residues and in which a very wide range of compoundsmay be accommodated. Three tentative binding sites for long-chainfatty acids, each with different surroundings, are located atthe surface of each domain.     

Soluble, prolonged-acting insulin derivatives. I. Degree of protraction and crystallizability of insulins substituted in the termini of the B-chain

Hydrophilic insulins, more positively charged than human insulinat neutral pH, have been prepared by substitution with basicamino acids at the termini of the B-chain and by blocking theC-terminal carboxyl group of the B-chain. The iso-electric pHof the insulin is thereby moved from 5.4 towards physiologicallevels. Slightly acid solutions of derivatives, in which chargehas been added in the C-terminus of the B-chain, have a prolongedaction in vivo, in particular if the carboxyl group is blocked.It is found that the prolong ed-acting hydrophilic insulinscrystallize instantly when the pH is adjusted to 7. The prolongedaction is ascribed to this readiness to crystallization combinedwith a low solubility, which may be further decreased by Increasedconcentration of zinc ions. Hydrophobic insulins have a prolongedaction independent of the site of substitution even if the derivativeis soluble at physiological pH. Some derivatives were preparedfrom porcine Insulin by tryptic transpeptidatlon. N-terminalB-chain substituted insulins were prepared by alkylation ofa biosynthetic single-chain insulin precursor, followed by tryptictranspeptidation rendering the double chain insulin derivative.The observed blood glucose lowering in the rabbits implies thatneither N- nor C-terminal B-chain substitution results in substantialdeterioration of biological potency. An index for the degreeof protraction based on the blood glucose data is used to comparethe insulins.     

A low resolution model for the interaction of G proteins with G protein-coupled receptors

A model is presented for the interaction between G proteinsand G protein-coupled receptors. The model is based on the factthat this interaction shows little specificity and thus conservedparts of the G proteins have to interact with conserved partsof the receptors. These parts are a conserved negative residuein the G protein, a fully conserved arginine in the receptorand a series of residues that are not conserved but always hydrophobiclike the hydrophobic side of the C-terminal helix of the G proteinand the hydrophobic side of a helix in the C-terminal domainof the receptor. Other, mainly cytosolic, factors determinethe specificity and regulation of this interaction. The relationbetween binding and activation will be shown. A large body ofexperimental evidence supports this model. Despite the factthat the model does not provide atomic resolution, it can beused to explain some experimental data that would otherwiseseem inexplicable, and it suggests experiments for its falsificationor verification.     

Use of site-directed mutagenesis to obtain isomorphous heavy-atom derivatives for protein crystallography: cysteine-containing mutants of phage T4 lysozyme

Five different cysteine-containing mutants of the lysozyme frombacteriopbage T4 were used to explore the feasibility of usingsite-directed mutagenesis to generate isomorphous heavy-atomderivatives for protein crystallography. Cysteines 54 and 97,present in wild-type lysozyme, can be readily reacted with mercuricion to produce an excellent isomorphous heavy-atom derivative.Mutants with an additional cysteine at position 86,146,153 or157, or with Cys 97 replaced by Val, were engineered by site-directedmutagenesis. The mutant lysozyme Thr 157 - Cys reacts with mercuricchloride to give an excellent new derivatve although Cys 157is only -60% substituted with the heavy atom. The cysteine atposition 146 is largely buried but reacts readily with mercuricchloride. In this case the isomorphism is poor and the resultantderivative is of marginal quality. Cys 153 reacts rapidly withmercuric ion but the derivative crystals do not diffract. Themutant Pro 86 - Cys does not yield a particularly good heavy-atomderivative. This is due in part to a loss of isomorphism associatedwith the mutation. In addition, Cys 86 shows very little reactivitytowards mercurials even though it is fully exposed to solvent.The mutation Cys 97 Val was used to explore the possibilityof creating an independent derivative by deleting a heavy-atomsite already present in wild-type lysozyme. In all cases thatwere tested, the quality of the heavy-atom derivative was improvedby using as an isomorphous pair mercury-substituted mutant versusnon-substituted mutant rather than mercury-substituted mutantversus (non-substituted) wild-type lysozyme. Unexpectedly, thecysteines that are most exposed to solvent and most mobile areleast reactive toward mercuric chloride. The cysteines thatprovide the best heavy-atom sites are those that are locatedin surface crevices and are only partly exposed to solvent.     

磺酸功能化离子液体催化甘油与甲醇醚化反应

考察了[HSO₃-bmim]CF₃SO₃、[HSO₃-bmim]P-TSA、[HSO₃-bmim]HSO₄和[HSO₃-bmim]H₂PO₄四种磺酸功能化离子液体对甘油与甲醇醚化反应的影响。结果表明,离子液体的催化性能与其酸强度相关联,[HSO₃-bmim]CF₃SO₃离子液体的酸强度最强,其催化性能也最好。以[HSO₃-bmim]CF₃SO₃为催化剂,在w([HSO₃-bmim]CF₃SO₃)/w(甘油)=0.5:1(质量比)、n(甲醇)/n(甘油)=8:1(摩尔比)、反应温度190℃、反应时间8 h时,甘油的转化率为84.5%,单甲基甘油醚的选择性为41.4%,二甲基甘油醚和三甲基甘油醚的联合选择性为34.1%。在此基础上,提出了离子液体[HSO₃-bmim]CF₃SO₃催化甘油与甲醇醚化反应的反应机理。     

Soluble, prolonged-acting insulin derivatives. II. Degree of protraction and crystallizability of insulins substituted in positions A17, B8, B13, B27 and B30

It has previously been found that insulins, to which positivecharge has been added by substitutions in position B30, thusraising the isolectric point towards pH 7, had a prolonged actionwhen injected as slightly acidic solutions because such derivativescrystallize very readily upon neutralization. Positive chargehas now been added by substituting the B13 and A17 glutainicacid residues with glutamines and B27 threonine with lysineor arginine. These substitutions were introduced by site-specificmutagenesis in a gene coding for a single-chain insulin precursor.By tryptic transpeptidation the single-chain precursors weretransformed to the double-chain insulin structure, concomitantlywith incorporation of residue B30. Thus insulins combining B13glutamine, A17 glutamine and B27 lysine or arginine with B30threonine, threonine amide or lysine amide were synthesized.The time course of blood glucose lowering effect and the absorptionwere studied after subcutaneous injection in rabbits and pigs.The prolonged action of B30-substituted insulins s marked lyenhanced by B27 lysine or arginine substitutions and by B13glutamine. The B27 residue is located on the surface of thehexamer, so a basic residue in this position presumably promotesthe packing of hexainers at neutral pH. The B13 residues clusterin the centre of the hexainer. When the electrostatic repulsiveforces from six glutamic acid residues are abolished by substitutionwith glutamine, a stabilization of the hexamer can he envisaged.The biological potency of insulins was measured in the freefat cell assay and in the mouse blood glucose assay test. Apotency factor could be fitted to each substitution, so thatthe potency of analogs with two or more substitutions can beestimated by multiplication of the corresponding potency factors.A charge-indifferent substitution, B8 glycine to serine, resultedin insulins that crystallize well but have low potencies. Alate elution in gradient r.p.-h.p.l.c. indicates that hydrophobicamino acid residues have been exposed as a result of this B8substitution. This most likely results from distortion of the-helix cominenc ing at residue B7, permitted only by B8 glycinewith dihedral angles (, ) of a D-amino acid.     

Soluble, prolonged-acting insulin derivatives. III. Degree of protraction, crystallizability and chemical stability of insulins substituted in positions A21, B13, B23, B27 and B30

It was previously demonstrated that insulins to which positivecharge has been added by substituting B13 glutamic acid witha glutamine residue, B27 threonine with an arginine or lysineresidue, and by blocking the C-terminal carboxyl group of theB-chain by amidation, featured a prolonged absorption from thesubcutis of rabbits and pigs after injection in solution atacidic pH. The phenomenon is ascribed to a low solubility combinedwith the readiness by which these analogs crystallize as theinjectant is being neutralized in the tissue. However, acidsolutions of insulin are chemically unstable as A21 asparagineboth deamidates to aspartic acid and takes part in formationof covalent dimers via -amino groups of other molecules. Inorder to circumvent the instability, substitutions were introducedin position A21, in addition to those in B13, B27 and B30, challengingthe fact that A21 asparagine has been conserved in this positionthroughout the evolution. Biological potency was retained whenglycine, serine, threonine, aspartic acid, histidine and argininewere introduced in this position, although to a varying degree.In the crystal structure of insulin a hydrogen bond bridgesthe -nitrogen of A21 with the backbone carbonyl of B23 glycine.In order to investigate the importance of this hydrogen bondfor biological activity a gene for the single-chain precursorB-chain(1-29)-Ala-Ala-Lys-A-chain(1-21) featuring an A21 prolinewas synthesized. However, this single-chain precursor failedto be properly produced by yeast, pointing to the formationof this hydrogen bond as an essential step in the folding process.The stability of the A21-substituted analogs in acid solutions(pH 3–4) with respect to deamidation and formation ofdimers was {small tilde}5–10 times higher than that ofhuman insulin in neutral solution. The rate of absorption ofmost insulins is decreased by increasing the Zn² concentrationof the preparation. However, one analog with A21 glycine showedfirst-order absorption kinetics in pigs with a half-life of{small tilde}25 h, independent of the Zn² concentration. Theday-to-day variation of the absorption of this analog was significantlylower than that of the conventional insulin suspensions, a propertythat might render such an insulin useful in the attempts toimprove glucose control in diabetics by a more predictable deliveryof basal insulin.     

手性药物开发战略的再认识

基础生物化学研究推动手性药物市场持续增长 ,2 0 0 1年单一对映体药物市场为 14 70亿美元 ,占世界药品市场 4 10 0亿美元的 36 %。预计 2 0 0 8年将突破 2 0 0 0亿美元大关 ,年增长率在 7%～ 8%。选择手性药物开发战略的重要依据是手性药理学研究结果 ,同时还应综合考虑科学和经济学标准。     

尿素甘油共塑化热塑性淀粉

采用尿素、甘油为塑化剂制备热塑性淀粉材料,并对改性后淀粉的力学性能、塑化效果以及结晶状况进行了探索,考察了尿素与甘油不同比例对热塑性淀粉塑化效果的影响,得出热塑性淀粉最佳塑化质量比为淀粉/尿素/甘油为100/20/20。在RH为33%的湿度环境保存1周,上述比例塑化的热塑性淀粉拉伸模量可达0.959 MPa,断裂伸长率达209.64%,且热塑性淀粉内未出现尿素自结晶,淀粉塑化效果良好。     

烷基甘油醚硫酸酯盐的合成

以甘油为原料制备了二辛基甘油醚硫酸盐阴离子表面活性剂,分别以IR和MS对中间产物二辛基甘油醚进行了结构表征。考察了物料摩尔比、反应温度、反应时间等因素对中间产物二辛基甘油醚收率的影响,得出了制备二辛基甘油醚的适宜工艺条件：辛醇钠与二氯丙醇摩尔比为2.2∶1,反应温度为70 ℃,反应时间为8 h。经GC内标法分析,上述条件下二辛基甘油醚的收率大于92%。以氯磺酸为硫酸化试剂,制得了二辛基甘油醚硫酸钠,并对其表面活性进行了测定,结果显示,该新型表面活性剂具有较好的降低表面张力的能力和较低的临界胶束浓度。     

The effect of experimental resolution on the performance of knowledge-based discriminatory functions for protein structure selection

The key to an accurate method of protein structure prediction is the development of an effective discriminatory function. Knowledge-based discriminatory functions extract parameters from statistical analysis of experimentally determined protein structures. We assess how the quality of the protein structures used for compiling statistics affects the performance of a residue-specific all-atom probability discriminatory function (RAPDF). We find that the discriminatory power correlates with the quality of the structural dataset on which the RAPDF is parameterized in a statistically significant manner. The overrepresentation of unfavorable contacts in the low-resolution and NMR structures contributes to the major errors in the compilation of the conditional probabilities. Such errors weaken the discriminatory power of the function, especially when decoy conformations also contain considerable numbers of unfavorable contacts. This indicates that using high-resolution structural datasets after filtering out unfavorable contacts can improve the performance of knowledge-based discriminatory functions.     

蔗糖高压催化氢化裂解制甘油

概述了我国甘油工业的发展状况及世界发展趋势。介绍了蔗糖催化氢解法制备甘油的工艺条件和影响氢解反应的因素。指出根据我国实际情况开展蔗糖氢解法制备甘油的研究，将为我国甘油工业的发展探索一条新路。