Effect of additives on the renaturation of reduced lysozyme in the presence of 4 M urea

Reduced lysozyme was renatured by sulfhydryl-disuffide interchangereactions at pH 8.0 in the presence of 4 M urea, with or withoutadditives at 40°C. In the absence of additives, the finalfolding yield of reduced lysozyme was 40%. In the presence ofsarcosine, glycerol, ammonium sulfate, N-acetyl glucosamineand glucose, its folding yields increased in all cases. In particular,yields increased up to 90% in the presence of 4 M sarcosine.On the other hand, the melting temperatures of lysozyme withor without additives in 0.02 M citrate buffer (pH 6.0) wereevaluated using differential scanning calorimetry. In the absenceof additive, the melting temperature of lysozyme was 73.8°C.In the presence of additives, all melting temperatures werehigher than that of lysozyme in the absence of additives. Moreover,there was a good correlation on addition of additives betweenan increase in the folding yield of reduced lysozyme with 4M urea and an increase in the melting temperature without 4M urea. Therefore, we conclude that additives, which stabilizenative lysozyme, are effective at increasing the folding yieldof reduced lysozyme in 4 M urea.     

The role of net charge on the renaturation of reduced lysozyme by the sulfhydryl-disulfide interchange reaction

Reduced and acetylated lysozymes are basic proteins. When theiramino groups were variously acetylated and then renatured bysulfhydryl-disulfide (SH-SS) interchange reaction at pH 8.0,the final folding yield decreased as the number of positivecharges decreased. The final folding yield of native and Ac1lysozyme, with one positive charge eliminated, was less sensitiveto increasing protein concentration than that of Ac2 lysozyme,where two positive charges had been eliminated. The final foldingyield of reduced Ac2 lysozyme increased in the presence of 1M urea, which reduced the aggregation of unfolded lysozyme.Thus, the aggregation of unfolded lysozymes, which leads toa decrease in the final folding yield, was found to be heavilydependent on their net charges. Moreover, the final foldingyield of reduced lysozyme was shown to be increased by use ofcystamine as an oxidizing reagent in comparison with 2-hydroxyethyldisulfide or dithiodiglycolic acid. This may support the ideathat the final folding yield is influenced by electrostaticinteraction between unfolded lysozymes in the early stage ofrenaturation. In contrast, the concentration dependency of thefinal folding yield of Ac1 lysozyme was different from thoseof carboxymethylated His15 and Asp106 lysozymes whose positivenet charges were similar to that of Ac1 lysozyme. On the basisof the observations, it is suggested that the formation of theaggregates in the renaturation process might also be affectedby the structure of the unfolded state of lysozyme in solution.     

Effective renaturation of denatured and reduced immunoglobulin G in vitro without assistance of chaperone

IgG is an oligomeric protein that consists of two heavy andtwo light chains. To form the oligomer, a highly concentratedprotein would be required on renaturation. On the other hand,refolding of proteins at high concentration often led to aggregation.Therefore, denatured and reduced oligomeric protein scarcelyrefolded to the native structure. As was expected, the foldingyield of the denatured and reduced IgG was below 5% under thecondition employed in rapid dilution. The low folding yieldwas elucidated to be due to assembly or aggregation. Using arenaturation method previously developed to depress aggregationeffectively by means of slow dialysis, the refolding yield ofthe denatured and reduced IgG at above 1 mg/ml was above 70%.Most of the refolded IgG was identical with the intact materialbased on analyses by affinity chromatography and SDS-PAGE.     

Point mutation of alanine (31) to valine prohibits the folding of reduced lysozyme by sulfhydryl--disulfide interchange

In the preceding paper in this issue, we described the overproduction of one mutant chicken lysozyme in Escherichia coil.Since this lysozyme contained two amino acid substitutions (Ala³¹ValandAsn¹⁰⁶Ser)in addition to an extra methionine residue at theNH₂-terminus the substituted amino acid residues were convertedback to the original ones by means of oligonucleotide-directedsite-specific mutagenesis and in vitro recombination. Thus fourkinds of chicken lysozyme [Met^–1 Val³¹Ser¹⁰⁶-, Met^–1Ser¹⁰⁶-,Met^–1 Val³¹-and Met^–1 (wild type)] wereexpressed in E. coli. From the results of folding experimentsof the reduced lysozymes by sulfhydryl-disulfide interchangeat pH 8.0 and 38°C, follow ed by the specific activity measurementsof the folded en zymes, the following conclusions can be drawn:(i) an extra methionine residue at the NH₂-terminus reducesthe folding rate but does not affect the lysozyme activity ofthe folded enzyme; (ii) the substitution of Asn¹⁰⁶ by Ser decreasesthe activity to 58% of that of intact native lysozyme withoutchanging the folding rate; and (iii) the substitution of Ala³¹Val prohibits the correct folding of lysozyme. Since the wildtype enzyme (Met^–1-lysozyme) was activated in vitro withoutloss of specific activity, the systems described in this study(mutagenesis, overproduction, purification and folding of inactivemutant lysozymes) may be useful in the study of folding pathways,expression of biological activity and stability of lysozyme.     

Engineering of human lysozyme as a polyelectrolyte by the alteration of molecular surface charge

The surface positive charges of human lysozyme were either increasedor decreased to alter the electrostatic interaction betweenenzyme and substrate in the lytic action of human lysozyme usingsite-directed mutagenesis. The amino acid substitutions accompanyingeither the addition or the removal of two units of positivecharge have shifted the optimal ionic strength (NaCl concentrationin 10 mM Mes buffer, pH 6.2) for the lysis of Micrococcus lysodeikticuscell from 0.04 M to 0.1 M and from 0.04 M to 0.02 M respectively.In addition to the change in ionic strength–activity profile,the pH–activity profile and the effect of a polycationicelectrolyte, poly-L-Lys-HCl, on the lytic activity were significantlychanged. Owing to the shifts in both ionic strength profilesand pH profiles the Arg⁷⁴/Arg¹²⁶ mutant has become a bettercatalyst than wild-type enzyme under the conditions of highionic strength and high pH, and the Gln⁴¹/Ser¹⁰¹ mutant hasbecome a better catalyst under the conditions of low ionic strengthand low pH.     

Development of an optimized refolding process for recombinant Ala-Glu-IGF-1

Denatured and reduced N-terminal extended insulin-like growthfactor-1 (AE-IGF-1) was purified from Escherichia coli extractsand subjected to in vitro folding. The renaturation processwas shown to be a function of the redox potential of the solution.Folding by different methods had no significant effect on therenaturation. A maximal yield of 60% (w/w)was obtained. Thefolded AE-IGF-1 was enzymatically converted to IGF-1. The majorby-product (20% w/w) was identified as scrambled IGF-1. Enzymaticdigestion at alkaline and acidic pH suggested two possible disulphidebond arrangements: (i) Cys⁶–Cys⁴⁷, Cys¹⁸–Cys⁶¹,Cys⁴⁸–Cys⁵²; or (if) Cys⁶–Cys⁵², Cys^l8–Cys⁶¹,Cys⁴⁷ and Cys⁴⁸ being in their reduced forms. Energy minimizationand molecular modelling suggested that the scrambled IGF-1,having reduced cysteines at positions 47 and 48, was the energeticallymost stable conformation of the two.     

Sulfolobus solfataricus protein disulphide oxidoreductase: insight into the roles of its redox sites

Sulfolobus solfataricus protein disulphide oxidoreductase (SsPDO)contains three disulphide bridges linking residues C⁴¹XXC⁴⁴,C¹⁵⁵XXC¹⁵⁸, C¹⁷³XXXXC¹⁷⁸. To get information on the role playedby these cross-links in determining the structural and functionalproperties of the protein, we performed site-directed mutagenesison Cys residues and investigated the changes in folding, stabilityand functional features of the mutants and analysed the resultswith computational analysis. The reductase activity of SsPDOand its mutants was evaluated by insulin and thioredoxin reductaseassays also coupled with peroxiredoxin Bcp1 of S. solfataricus.The three-dimensional model of SsPDO was constructed and correlatedwith circular dichroism data and functional results. Biochemicalanalysis indicated a key function for the redox site constitutedby Cys155 and Cys158. To discriminate between the role of thetwo cysteine residues, each cysteine was mutagenised and thebehaviour of the single mutants was investigated elucidatingthe basis of the electron-shuffling mechanism for SsPDO. Finally,cysteine pK values were calculated and the accessible surfacefor the cysteine side chains in the reduced form was measured,showing higher reactivity and solvent exposure for Cys155.     

Engineered turns of a recombinant antibody improve its in vivo folding

Using recombinant antibodies functionally expressed by secretionto the periplasm in Escherichia coli as a model system, we identifiedmutations located in turns of the protein which reduce the formationof aggregates during in vivo folding or which influence cellstability during expression. Unexpectedly, the two effects arebased on different mutations and could be separated, but bothmutations act synergistically in vivo. Neither mutation increasesthe thermodynamic stability in vitro. However, the in vivo foldingmutation correlates with the yield of oxidative folding in vitro,which is limited by the side reaction of aggregation. The invivo folding data also correlate with the rate and activationentropy of thermally induced aggregation. This analysis showsthat it is possible to engineer improved frameworks for semi-syntheticantibody libraries which may be important in maintaining librarydiversity. Moreover, limitations in recombinant protein expressioncan be overcome by single amino acid substitutions.     

高浓度变性-还原溶菌酶的流加复性动力学特性

研究了高浓度变性-还原溶菌酶的流加复性过程动力学特性,重点考察了影响复性速率和复性收率的主要因素,包括盐酸胍浓度、酶浓度和氧化还原剂浓度.结果表明,与直接稀释复性相比,流加操作可有效降低肽链分子间聚集,提高蛋白质的复性收率;在流加复性条件下,随着酶浓度的提高,复性速率和复性收率均有所下降,但适当提高复性液中盐酸胍浓度仍可获得高浓度蛋白质的高复性收率.另外,随着变性酶浓度的提高,需要适当提高复性液中氧化型谷胱甘肽浓度,以加快溶菌酶分子内二硫键的形成,提高复性反应速度.     

Lysozyme requires fluctuation of the active site for the manifestation of activity

Mutations around His15 which lie far away from the active site,stimulated glycol chitin activity of lysozyme at physiologicaltemperature. Del-Argl4Hisl5 lysozyme, a mutant lysozyme whoseArgl4 and Hisl5 were deleted together, and has the highest activityamong these mutant lysozymes, had a similar binding abilityto a trimer of N-acetyl-glucosamine, a substrate analogue, relativeto native lysozyme. This suggests that the increased activitywas due to an increased k_cat in the catalysis reaction. TheH-D exchange rate of the N-1 proton in the Trp63 which is locatedin the active site cleft, was enhanced in the Del-Argl4Hisl5lysozyme, while 2-D proton NMR analysis revealed no conformationalchange around Trp63. We conclude that some sort of fluctuationat the active site might be required for the manifestation ofactivity. This theory is supported by the finding that the Del-Argl4Hisl5lysozyme showed a shift in temperature dependency of activityto lower temperatures compared with that of native lysozyme.     

Secretion and in vivo folding of the Fab fragment of the antibody McPC603 in Escherichia coli: influence of disulphides and cis-prolines

Using the well-characterized antibody McPC603 as a model, wehad found that the F_v fragment can be isolated from Escherichiacoli as a functional protein in good yields, whereas the amountof the correctly folded F_ab fragment of the same antibody producedunder identical conditions is significantly lower. In this paper,we analyse the reasons for this difference. We found that avariety of signal sequences function in the secretion of theisolated chains of the F_ab fragment or in the co-secretion ofboth chains in E.coli. The low yield of functional F_ab fragmentis not caused by inefficient expression or secretion in E.coli,but by inefficient folding and/or assembly in the periplasm.We compared the folding yields for the F_v and the F_ab fragmentin the periplasm under various conditions. Several diagnosticframework variants were constructed and their folding yieldsmeasured. The results show that substitutions affecting cis-prolineresidues and those affecting various disulphide bonds in theprotein are by themselves insufficient to dramatically changethe partitioning of the folding pathway to the native structure,and the cause must lie in a facile aggregation of folding intermediatescommon to all structural variants. However, all structural variantscould be obtained in native form, demonstrating the generalutility of the secretory expression strategy.     

重组人γ-干扰素包涵体稀释复性

采用稀释法研究了重组人γ-干扰素包涵体复性条件,如温度、pH值、蛋白浓度、复性液中脲浓度,同时对加样操作方式进行了考察,得到了较为适宜的复性条件.结果表明,脲能有效地抑制复性过程中蛋白质的聚集,合适的脲浓度随复性蛋白浓度的增加而有所增大,在4℃、pH值8.0、脲浓度1.0mol•L^-1、蛋白浓度0.1mg•ml^-1及脉冲加样操作方式等条件下,复性后重组人γ-干扰素的活性为2.39×10⁵ IU•ml^-1,比活达2.21×10⁷ IU•mg^-1.     

Analysis of a catalytic pathway via a covalent adduct of D52E hen egg white mutant lysozyme by further mutation

We previously demonstrated by X-ray crystallography and electrospraymass spectrometry that D52E mutant hen lysozyme formed a covalentenzyme–substrate adduct on reaction with N-acetylglucosamineoligomer. This observation indicates that D52E lysozyme mayacquire a catalytic pathway via a covalent adduct. To explainthis pathway, the formation and hydrolysis reactions of thecovalent adduct were investigated. Kinetic analysis indicatedthat the hydrolysis step was the rate-limiting step, 60-foldslower than the formation reaction. In the formation reaction,the pH dependence was bell-shaped, which was plausibly explainedby the functions of the two catalytic pK_as of Glu35 and Glu52.On the other hand, the pH dependence in the hydrolysis was sigmoidalwith a transition at pH 4.5, which was identical with the experimentallydetermined pK_a of Glu35 in the covalent adduct, indicating thatGlu35 functions as a general base to hydrolyze the adduct. Toimprove the turnover rate of D52E lysozyme, the mutation ofN46D was designed and introduced to D52E lysozyme. This mutationreduced the activation energy in the hydrolysis reaction ofthe covalent adduct by 1.8 kcal/mol at pH 5.0 and 40°C butdid not affect the formation reaction. Our data may providea useful approach to understanding the precise mechanism ofthe function of natural glycosidases, which catalyze via a covalentadduct.     

Effects of signal peptide changes on the secretion of bovine somatotropin (bST) from Escherichia coli

Bovine somatotropin (bST) was secreted from Escherichia coliat moderate levels of 1–2 µg/ml/OD using expressionvectors in which the bST gene was fused to the lamB secretionsignal. To study the secretion properties of bST in E.coli further,two approaches for modifying the secretion signal were employed.In the first case, fusion proteins were constructed with sixalternative bacterial secretion signals: three from E.coli proteins(HisJ, MalE and OmpA), two from bacteriophage proteins (M13coat protein and PA-2 Lc) and one from the chitinase A proteinof Serratia marcescens. The results, as monitored by Westernblot analysis of both total cell protein and the periplasmicfraction, showed that these changes in the secretion signaldid not significantly affect the secretion properties of bST.In the second approach, a library of random mutations was createdin the lamB secretion signal and 200 independent clones werescreened. The level of secreted bST was determined by growingindividual clones in duplicate in microtiter wells, inducingprotein expression and measuring the bST released by osmoticshock using a particle concentration fluorescent immunoassay.The secretion properties of several novel variants in the LamBsignal peptide are presented.     

A novel dialysis procedure for the crystallization of proteins

Various dialysis methods are commonly employed for the crystallizationof proteins. Typical procedures include the use of dialysisbags, dialysis buttons or Zeppezauer microdiffusion cells. Thegeneral principle involved is that the protein solution is graduallybrought to a point of supersaturation by imposing a gradientof ionic strength or organic solvent concentration across thewall of the dialysis membrane. However, in some cases, the impositionof this gradient across the dialysis membrane can result inthe formation of a large number of crystal nucleation sites,thereby giving rise to a reduction in the maximum size of thecrystals which can be obtained. A novel ‘double-dialysis’procedure which incorporates a second dialysis membrane, thusreducing the rate of equilibration in the crystallization experiment,has been developed in our laboratory. The system has been employedsuccessfully on the delta toxin of Staphylococcus aureus resultingin a useful increase in crystal size. A more quantitative analysisof the technique has been carried out on rat liver malic enzyme.The results of a limited series of crystallization trials withthis protein have shown that employment of the ‘double-dialysis’technique allows a fine control of the rate of crystal nucleationand therefore provides a mechanism for the controlled growthof large crystals.     

Stabilization of lysozyme against irreversible inactivation by alterations of the Asp-Gly sequences

Site-directed mutagenesis was performed at Asp-Gly (48–49,66–67, 101–102) and Asn-Gly (103–104) sequencesof hen egg-white lysozyme to protect the enzyme against irreversiblethermoinactivation. Because the lysozyme inactivation was causedby the accumulation of multiple chemical reactions, includingthe isomerization of the Asp-Gly sequence and the deamidationof Asn [Tomizawa et al.(1994) Biochemistry, 33, 13032–13037],the suppression of these reactions by the substitution of Glyto Ala, or the introduction of a sequence of human-type lysozyme,was attempted and the mutants (where each or all labile sequenceswere replaced) were prepared. The substitution resulted in thereversible destabilization from 1 to 2 kcal/mol per substitution.The destabilization was caused by the introduction of ß-carbonto the constrained position that had conformational angles withinthe allowed range for the Gly residue. Despite the decreasein the reversible conformational stability, the mutants hadmore resistance to irreversible inactivation at pH 4 and 100°C.In particular, the rate of irreversible inactivation of themutant, which was replaced at four chemically labile sequences,was the latest and corresponded to 18 kcal/mol of the reversibleconformational stability. Therefore, replacement of the chemicallylabile sequence was found to be more effective at protectingenzymes against irreversible thermoinactivation than at strengtheningreversible conformational stability.     

The structure-function relationship of the lipases from Pseudomonas aeruginosa and Bacillus subtilis

Within the BRIDGE T-project on lipases we investigate the structure-functionrelationships of the lipases from Bacillus subtilis and Pseudomonasaeruginosa. Construction of an overproducing Bacillus. strainallowed the purification of > 100 mg lipase from 30 l culturesupernatant. After testing a large variety of crystallizationconditions, the Bacillus lipase gave crystals of reasonablequality in PEG-4000 (38-45%), Na₂SO₄ and octyl-ß-glucosideat 22°C, pH 9.0. A 2.5 Å; dataset has been obtainedwhich is complete from 15 to 2.5 A resolution. P.aeruginosawild-type strain PAC1R was fermented using conditions of maximumlipase production. More than 90% of the lipase was cell boundand could be solubilized by treatment of the cells with TritonX-100. This permitted the purification of 50 mg lipase. So far,no crystals of sufficient quality were obtained. Comparisonof the model we built for the Pseudomonas lipase, on the basisof sequences and structures of various hydrolases which werefound to possess a common folding pattern (/ß hydrolasefold), with the X-ray structure of the P.glumae lipase revealedthat it is possible to correctly build the structure of thecore of a protein even in the absence of obvious sequence homologywith a protein of known 3-D structure.     

The hierarchy of mutations influencing the folding of antibody domains in Escherichia coli

In a systematic study of the periplasmic folding of antibodyfragments in Escherichia coli, we have analysed the expressionof an aggregation-prone and previously non-functional anti-phosphorylcholineantibody, T15, as a model system and converted it to a functionalmolecule. Introduction of heavy chain framework mutations previouslyfound to improve the folding of a related antibody led to improvedfolding of T15 fragments and improved physiology of the hostE.coli cells. Manipulation of the complementarity determiningregions (CDR) of the framework-mutated forms of T15 furtherimproved folding and bacterial host physiology, but no improvementwas seen in the wild type, suggesting the existence of a hierarchyin sequence positions leading to aggregation. Rational mutagenesisof the T15 light chain led to the production of functional T15fragments for the first time, with increased levels of functionalprotein produced from V_H manipulated constructs. We proposethat a hierarchical analysis of the primary amino acid sequence,as we have described, provides guidelines on how correctly folding,functional antibodies might be achieved and will allow furtherdelineation of the decisive structural factors and pathwaysfavouring protein aggregation.     

Recombinant pro-regions from papain and papaya proteinase IV are selective high affinity inhibitors of the mature papaya enzymes

Proteolytic enzymes require the presence of their proregionsfor correct folding. Of the four proteolytic enzymes from Caricapapaya, papain and papaya proteinase IV (PPIV) have 68% sequenceidentity. We find that their proregions are even more similar,exhibiting 73.6% identity. cDNAs encoding the pro-regions ofthese two proteinases have been expressed in Escherichia coliindependently from their mature enzymes. The recombinant pro-regionsof papain and PPIV have been shown to be high affinity inhibitorsof all four of the mature native papaya cysteine proteinases.Their inhibition constants are in the range 10^–6–;10^–;9M. PPIV was inhibited two to three orders of magnitude lesseffectively than papain, chymopapain and caricain. The pro-regionof PPIV, however, inhibited its own mature enzyme more effectivelythan did the proregion of papain. Alignment of the sequencesof the four papaya enzymes shows that there is a highly variablesection towards the C-terminal of the pro-region. This regionmay therefore confer selectivity to the pro-regions for theindividual proteolytic enzymes.     

Determination of the pKa values of titratable groups of an antigen-antibody complex, HyHEL-5-hen egg lysozyme

The titration behavior of the ionizable residues of the HyHEL-5–henegg lysozyme complex and its individual components has beenstudied using continuum electrostatic calculations. Severalresidues of HyHEL-5 had pK_a values shifted away from model valuesfor isolated residues by more than three pH units. Shifts awayfrom the model values were smaller for the residues of hen egglysozyme. A moderate variation in the pK_a values of the titratablegroups was observed upon increase of the ionic strength from0 to 100 mM, amounting to 1–2 pH units in most cases.Under physiological conditions, the net charge of HyHEL-5 wasopposite that for hen egg lysozyme. Several residues, includingthose involved in the Arg–Glu salt bridges that have beenproposed to be important in antibody-antigen binding, had pK_avalues that were changed significantly upon binding. The maintitration event upon antibody-antigen binding appears to beloss of a proton from residue GluH50 of the Fv molecule. Thelimitations of our calculation methods and the role they mightplay in the design of antibodies for use in assays, sensorsand separations are discussed