High-level expression, purification, kinetic characterization and crystallization of protein farnesyltransferase -subunit C-terminal mutants

Protein farnesyltransferase (FPT) is a 97 000 Da heterodimericenzyme that catalyzes post-translational farnesylation of manycellular regulatory proteins including p21 Ras. To facilitatethe construction of site-directed mutants, a novel translationallycoupled, two-cistron Escherichia coli expression system forrat FPT has been developed. This expression system enabled yieldsof >5 mg of purified protein per liter of E.coli cultureto be obtained. The E.coli-derived FPT demonstrated an activitycomparable to that of protein isolated from other sources. Thereported expression system was used to construct three ß-subunitC-terminal truncation mutants, 5, 10 and 14, which were designedto eliminate a lattice interaction between the ß-subunitC-terminus of one molecule and the active site of a symmetry-relatedmolecule. Steady-state kinetic analyses of these mutants showedthat deletion up to 14 residues at the C-terminus did not reducethe value of k_cat; however, K_m values for both peptide and FPPincreased 2–3-fold. A new crystalline form of FPT was obtainedfor the 10 C-terminal mutant grown in the presence of the substrateanalogs acetyl-Cys-Val-Ile-Met-COOH peptide and -hydroxyfarnesylphosphonicacid. The crystals diffract to beyond 2.0 Å resolution.The refined structure clearly shows that both substrate analogsadopt extended conformations within the FPT active site cavity.     

Cloning, expression and purification of a sarcoplasmic calcium-binding protein from the sandworm Nereis diversicolor via a fusion product with chloramphenicol acetyltransferase

A gene coding for the Nereis sarcoplasmic calcium-binding protein(NSCP) was synthesized and expressed in Escherichia coli. Thesequence of the gene was derived from the protein sequence byreverse translation. It possesses a number of unique, regularlyspaced, restriction endonuclease cleavage sites to facilitatefuture site-directed mutagenesis. For the cloning strategy thegene sequence was divided into four parts. Three parts werecloned by ligation of hybridized oligomers and one part by inversePCR. The protein was expressed as a fusion protein with thebacterial chloramphenicol acetyltransferase (CAT), which couldbe easily purified by affinity chromatography. At the junctionof the CAT and NSCP moieties a recognition site for the proteolyticenzyme factor Xa was built in. However, the distance betweenthe moieties appeared to be crucial to warrant cleavage. A kineticanalysis showed that NSCP prepared from the sandworm and theone expressed by E.coli behaved in the same way. This systemprovides a basis for site-specific mutagenesis studies, in orderto elucidate the molecular mechanism of cation binding and concomitantconformational changes     

Engineering ribonuclease A: production, purification and characterization of wild-type enzyme and mutants at Gln11

Bovine pancreatic ribonuclease A (RNase A) has been the objectof much landmark work in biological chemistry. Yet the applicationof the techniques of protein engineering to RNase A has beenlimited by problems inherent in the isolation and heterologousexpression of its gene. A cDNA library was prepared from cowpancreas, and from this library the cDNA that codes for RNaseA was isolated. This cDNA was inserted into expression plasmidsthat then directed the production of RNase A in Saccharomycescerevisiae (fused to a modified -factor leader sequence) orEscherichia coli (fused to the pelB signal sequence). RNaseA secreted into the medium by S.cerevisiae was an active buthighly glycosylated enzyme that was recoverable at 1 mg/l ofculture. RNase A produced by E.coli was in an insoluble fractionof the cell lysate. Oxidation of the reduced and denatured proteinproduced active enzyme which was isolated at 50 mg/l of culture.The bacterial expression system is ideal for the large-scaleproduction of mutants of RNase A. This system was used to substitutealanine, asparagine or histidine for Gln11, a conserved residuethat donates a hydrogen bond to the reactive phosphoryl groupof bound substrate. Analysis of the binding and turnover ofnatural and synthetic substrates by the wild-type and mutantenzymes shows that the primary role of Gln11 is to prevent thenon-productive binding of substrate.     

Structure-function analysis of human IL-1{alpha}: identification of residues required for binding to the human type I IL-1 receptor

Using oligonucleotide-directed mutagenesis, the binding siteon human interleukin-1 (IL-1) for the human type I IL-1 receptor(IL-1R) has been analyzed. Substitution of seven amino acids(Arg12, Ile14, Asp60, Asp61, Ile64, Lys96 and Trp109) resultedin a significant loss of binding to the receptor. Based on crystallographicinformation, the side chains of these residues are clusteredin one region of IL-1 and exposed on the surface of the protein.Five of the residues in the IL-1 binding site align with thebinding residues previously determined in human IL-1ß,demonstrating that the type I IL-1R recognizes homologous regionsin both ligands. Unexpectedly, only three of the aligned residuesare identical between IL-1 and IL-1ß. These observationssuggest that the composition of contact residues in the bindingsite is unique for each ligand–receptor complex in theIL-1 system.     

Expression, purification and characterization of recombinant crambin

Crambin, a small hydrophobic protein (4.7 kDa and 46 residues),has been successfully expressed in Escherichia coli from anartificial, synthetic gene. Several expression systems wereinvestigated. Ultimately, crambin was successfully expressedas a fusion protein with the maltose binding protein, whichwas purified by affinity chromatography. Crambin expressed asa C-terminal domain was then cleaved from the fusion proteinwith Factor Xa protease and purified. Circular dichroism spectroscopyand amino acid analysis suggested that the purified materialwas identical to crambin isolated from seed. For positive identificationthe protein was crystallized from an ethanol–water solution,by a novel method involving the inclusion of phospholipids inthe crystallization buffer, and then subjected to crystallographicanalysis, Diffraction data were collected at the Brookhavensynchrotron (beamline-X12C) to a resolution of 1.32 Åat 150 K. The structure, refined to an R value of 9.6%, confirmedthat the cloned protein was crambin. The availability of clonedcrambin will allow site-specific mutagenesis studies to be performedon the protein known to the highest resolution.     

The role of asparagine-32 in forming the receptor-binding epitope of human epidermal growth factor

The highly conserved asparagine residue at position 32 (Asn32)in the 'hinge' region of epidermal growth factor (EGF) separatesthe N- and C-terminal structural motifs of the EGF moleculeand is therefore an appropriate target for structure-functionstudies. Analogs of human EGF (hEGF) were generated in whichAsn32 was substituted with aspartate, glycine, isoleucine, lysine,proUne and tryptophan. The relative affinity of the EGF receptorfor mutant hEGF analogs was determined by radioreceptor competitionassay. A wide range of receptor affinities was observed dependingon the amino acid substitution. N32K and N32W hEGF analogs hadrelatively high receptor affinity, while the N32G and N32D analogsshowed decreased affinity, 35% and 25% respectively, relativeto wild type hEGF. However, no binding of the N32P analog wasdetected by radioreceptor competition assay. The N32P mutantdisplayed an NMR spectrum significantly different from thatof native wild type hEGF, indicating gross structural perturbation.In contrast, the N32K and N32D analogs exhibited spectra similarto that of native wild type hEGF. Genetically combining theN32D hEGF with an hEGF species having either the mutation L26Ghi the N-terminal region or L47A in the C-terminal region, generateddouble-mutant hEGF species whkh had relative affinities essentiallyequal to the product of the relative affinities of the parenthEGF mutants, indicating functionally independent changes inUgand-receptor interaction. These studies indicate the requirementfor H-bond donor functionality in the side chain of residuenumber 32 in forming a fully competent receptor-binding epitope.     

Large-scale expression, purification and characterization of small fragments of thrombomodulin: the roles of the sixth domain and of methionine 388

Fragments of human thrombomodulin (TM) have been expressed inlarge quantities in the Pichia pastoris yeast expression systemand purified to homogeneity. Fermentation of P.pastoris resultedin yields of 170 mg/1 TM. Purification to homogeneity resultedin an overall 10% yield, so that quantities of –20 mgpurified fragments can be readily obtained. Smaller fragmentsof TM, such as the individual fourth or fifth domains, werenot active, nor were equimolar mixtures of the two domains.These results demonstrate that the fourth and fifth epidermalgrowth factor (EGF)–like domains together comprise thesmallest active fragment of TM. The fragment containing thefourth and fifth EGFlike domains (TMEGF(4–5)] had 10%the specific activity of rabbit TM. Comparison of the M388Lmutant TMEGF(4–5) fragment with the same mutant TMEGF(4–5–6)fragment showed that the fragment with the sixth domain hada 10–fold better Km value for thrombin than the fragmentthat did not contain the sixth domain; this factor completelyaccounts for the higher specific activity of the fragments containingthe sixth domain. Comparison of the wild–type and M388Lmutants showed that the M388L mutation resulted in a 2–foldincrease in k_cat for the activation of protein C by the thrombin–TMfragment complex, completely accounting for the 2–foldincrease in specific activity of these mutant fragments.     

Lipid-tagged antibodies: bacterial expression and characterization of a lipoprotein--single-chain antibody fusion protein

In order to achieve a stable and functional immobilization ofantibodies, we investigated the possibility of adding hydrophobicmembrane anchors to antibody fragments expressed in Escherichiacoli. The DNA sequence encoding the signal peptide and the nineN-terminal amino add residues of the major lipoprotein of E.coliwas fused to the sequence of an anti-2-phenyloxazolone single-chainFv antibody fragment [Takkinen et al. (1991) Protein Engng,4, 837–841]. The expression of the fusion construct inE.coli resulted in specific accumulation of an immunoreactive28 kDa polypeptide. Unlike the unmodified single-chain Fv fragment,the fusion protein was cell-associated, labelled by [³H]palmitatewhich is indicative of the presence of N-terminal lipid modification,partitioned into the detergent phase upon Triton X-114 phaseseparation and was localized predominantly in the bacterialouter membrane. The fusion antibody displayed specific 2-phenyloxazolone-bindingactivity in the membranebound form and after solubilizationwith non-ionic detergents. Furthermore, upon removal of detergentthe fusion antibody was incorporated into proteoliposomes whichdisplayed specific hapten-binding activity. Our results showthat antibodies can be converted to membrane-bound proteinswith retention of antigen-binding properties by introductionof lipid anchors during biosynthesis. This approach may proveuseful in the design of immunoliposomes and immunosensors.     

Aspartic acid 50 and tyrosine 108 are essential for receptor binding and cytotoxic activity of tumour necrosis factor beta (lymphotoxin)

Single amino acid substitutions were generated in predictedhydrophilic loop regions of the human tumour necrosis factorbeta (TNF-ß) molecule, and the mutant proteins wereexpressed in Escherichia coli and purified. Mutants with singleamino acid changes at either of two distinct loop regions, atpositions aspartic acid 50 or tyrosine 108, were found to havegreatly reduced receptor binding and cytotoxic activity. Thesetwo regions in TNF-ß correspond to known loop regionswhere mutations also result in loss of biological activity ofTNF–, a related cytokine which shares the same cellularreceptors with TNF-ß. The two distinct loops at positions31-34 and 84-89 in the known three-dimensional structure ofTNF- (equivalent to positions 46–50 and 105–110respectively in TNF-ß), lie on opposite sides of theTNF- monomer. When the TNF-a monomer forms a trimer, the twoloops, each from a different subunit of the trimer, come togetherand lie in a cleft between adjacent subunits. Together, thesefindings suggest that a TNF receptor binds to a cleft betweensubunits via surface loops at amino acid residues 31–34and 84–89 in TNF–, and similarly via surface loopsincluding amino acids aspartic acid 50 and tyrosine 108 in TNF–ß.     

Engineering the C-region of human insulin-like growth factor-1: implications for receptor binding

Recombinant wild-type human IGF-1 and a C-region mutant in whichresidues 28–37 have been replaced by a 4-glycine bridge(4-Gly IGF-1) were secreted and purified from yeast. An IGF-1analogue in which residues 29–41 of the C-region havebeen deleted (mini IGF-1) was created by site-directed mutagenesisand also expressed. All three proteins adopted the insulin-foldas determined by circular dichroism. The significantly raisedexpression levels of mini IGF-1 allowed the recording of two-dimensionalNMR spectra. The affinity of 4-Gly IGF-1 for the IGF-1 receptorwas {small tilde}100-fold lower than that of wild-type IGF-1and the affinity for the insulin receptor was {small tilde}10-foldlower. Mini IGF-1 showed no affinity for either receptor. Notonly does the C-region of IGF-1 contribute directly to thefree energy of binding to the IGF-1 receptor, but also the absenceof flexibility in this region eliminates binding altogether.As postulated for the binding of insulin to its own receptor,it is proposed that binding of IGF-1 to the IGF-1 receptor alsoinvolves a conformational change in which the C-terminal B-regionresidues detach from the body of the molecule to expose theunderlying A-region residues.     

A novel yeast expression/secretion system for the recombinant plant thiol endoprotease propapain

A new high-yield yeast expression/secretion system has beenadapted for the plant thiol endoprotease papain. The propapaingene, obtained from Carica papaya fruit, is expressed in theyeast Saccharomyces cerevisiae. The gene was cloned into a FLAGepitope-tagging expression vector downstream of the yeast alphamating factor (-factor) secretion signal sequence. Expressionof the heterologous propapain in yeast is controlled by theglucose-repressible alcohol dehydrogenase isoenzyme II promoter(ADH2). Glycosylated FLAG-tagged propapain is secreted by asocalled ‘super secretor’ strain, pmr1 (ssc1), intothe culture supernatant where it accumulates to {small tilde}1.7mg/1. The proregion contains three consensus N-linked glycosylationsites, whereas there are only two such sites in previously reportedcDNA sequences. Removal of this third N-linked glycosylationsite results in a drastic reduction in the level of proteaseactivity present in the culture supernatant. Two different typesof affinity chromatography were used to purify either propapainor papain. The propapain precursor is autoproteolytically activatedto mature papain (M_r = 24 kDa) using conditions reported previously.The kinetic parameters obtained agree well with the literaturevalues. The yields of active papain are 10-fold higher thanthose previously reported for propapain in other yeast or bacterialexpression systems. This, together with the ease with whichmutant proteins can be made, makes this yeast advantageous fora structure–function analysis of recombinant wild-typeand mutant papain, and possibly for other related cysteine proteasesas well.     

Involvement of aspartic and glutamic residues in kringle-2 of tissue-type plasminogen activator in lysine binding, fibrin binding and stimulation of activity as revealed by chemical modification and oligonucleotide-directed mutagenesis

Modification of glutamic and aspartic acid residues of tissue-typeplasminogen activator (t-PA) with 1-ethyl-3(3-dimethyl-aminopropyl)-carbodiimideleads to a decrease in affinity for lysine and fibrin, to adecrease of plasminogen activation activity in the presenceof a fibrin mimic, but leaves amidolytic activity and plasminogenactivation without fibrin mimic unaffected. Experiments withkringle-2 ligands and a deletion mutant of t-PA (K₂P) suggeststhat glutamic or aspartic acid residues in K₂ of t-PA are involvedin stimulation of activity, lysine binding and fibrin binding.Mutant t-PA molecules were constructed by site-directed mutagenesisin which one or two of the five aspartic or glutamic acid residuesin K₂ were changed to asparagine or glutamine respectively.Mutation of Asp236 and/or Asp238 leads to t-PA molecules with3- to 4-fold lower specific activity in the presence of fibrinmimic and having no detectable affinity for lysine analogs.However, fibrin binding was not influenced. Mutation of Glu254also leads to a 3- to 4-fold lower activity, but to a much smallerreduction of lysine or fibrin binding. Residues Asp236 and Asp238are both essential for binding to lysine derivatives, whileGlu254 might be involved but is not essential. Residues Asp236,Asp238 and Glu254 are all three involved in stimulation of activity.Remarkably, mutation of residues Asp236 and/or Asp238 appearsnot to influence fibrin binding of t-PA whereas that of Glu254does.     

The S variant of human {alpha}1-antitrypsin, structure and implications for function and metabolism

The S variant of the human ₁-antitrysin with E-264 – V,is responsible for a mild ₁-antitrypsin deficiency quite commonin the European population. S protein specifically cleaved atthe susceptible peptide bond was crystallized and its crystalstructure determined and refined to 3.1 Å resolution.The S variant crystallizes isomorphous to the normal M variant.The difference Fourier electron density map shows the E –V change as outstanding residual density. In addition, smallstructural changes of the main polypeptide chain radiate fromthe site of mutation and affect parts far removed from it. Bythe mutation, internal hydrogen bonds and salt linkages of E-264to Y-38 and K-487, respectively, are lost. They cause the far-reachingslight distortions and are probobly related to the reduced thermalstability of the S mutant. They may also be responsible forslower folding of the polypeptide chain and the clinical symptomsof ₁-antitrypsin deficiency. In a theoretical study by moleculardynamics methods simulations of the M and S proteins were madeand the results analysed with respect to structrual and dynamicproperties and compared with the experimental results. Thereis a significant correlation between experimental and theoreticalresults in some respects.     

Expression and characterization of apolipoprotein(a) kringle IV types 1, 2 and 10 in mammalian cells

We have designed expression constructs containing sequencescorresponding to apolipoprotein(a) kringle FV types 1, 2 and10 and used these constructs to transfect human embryonic kidneycells. We have also expressed a mutant form of kringle FV type2 in which the N-linked glycosylation site has been removedby replacement of an asparagine residue with an alanine. Immunoprecipitationanalysis of [³⁵S]Cys-labeled transfected cell culture supernatantsresulted in the observation of two bands for kringle IV type1 (M_r 30 000 and 26 000), two bands for kringle IV type 2 (M_r25 000 and 22 000), two bands for kringle IV type 10 (M_r 27000 and 23 000) and one band for the glycosylation mutant (M_r22 000). In all cases, observed molecular weights greatly exceededthose predicted from amino acid sequence, suggesting the presenceof both N- and O-linked glycans. None of the recombinant singlekringles were observed to bind to fibrinogen as determined byELISA or by co-immunoprecipitation in the case of kringle IVtype 10 and only kringle IV type 10 was able to bind to lysine-Sepharose.These data suggest that apo(a) binding to fibrinogen/fibrinmay require motif(s) in addition to apo(a) kringle IV type 10.     

Structural and functional domains in human tumour necrosis factors

Human tumour necrosis factors (hTNFs) and ß are relatedpleiotropic cytokines which share many activities and competewith each other for binding to two receptor components on manycell types. Although structural and biological data indicatethat the active form of hTNF- may be a symmetrical trimer, themanner in which hTNFs interact with their receptors to triggera myriad of cell type-dependent responses is not clear. A combinationof chemical modification, epitope mapping and site-directedmutagenesis approaches suggest that at least four distinct peptidesequences are Important for the biological activity of hTNF-.In particular, certain peptide sequences between amino acidpositions 11 and 35 in hTNF- appear to be critical for receptorbinding and triggering biological responses. The recent cloningof the two hTNF-/ß receptors opens the way for precisemapping of the functional domains in hTNFs     

Mutational analysis of structure--activity relationships in human tumor necrosis factor-alpha

To determine the region of human tumor necrosis factor-alpha(TNF-), essential for cytotoxic activity against mouse L-M cells,single amino-acid-substituted TNF- mutant proteins (muteins)were produced in Escherichia coli by protein engineering techniques.An expression plasmid for TNF- was mutagenized by passage throughan E.coli mutD5 mutator strain and by oligonucleotide-directedmutagenesis. Approximately 100 single amino-acid-substitutedTNF- muteins were produced and assayed for cytotoxic activity.The cytotoxic activities of purified TNF- muteins, e.g. TNF-31T,-32Y, -82D, -85H, -115L, -141Y, -144K and -146E, were < 1%of that of parent TNF-. These results indicate that the integrityof at least four distinct regions of the TNF- molecule is requiredfor full biological activity. These regions are designated asfollows: region I, from position 30 to 32; region II, from position82 to 89; region III, from position 115 to 117; region FV, fromposition 141 to 146. In addition, TNF-141Y could not completelycompete with parent TNF- for binding to the receptor. This demonstratesthat region IV, and at least aspartk acid at position 141, mustbe involved in the TNF receptor binding site.     

In vivo copper- and cadmium-binding ability of mammalian metallothionein domain

The ß domain of mouse metallothionein 1 (ßMT) wassynthesized in Escherichia coli cells grown in the presenceof copper or cadmium. Homogenous preparations of Cu–ßMTand Cd–ßMT were used to characterize the correspondingin vivo-conformed metal-clusters, and to compare them with thespecies obtained in vitro by metal replacement to a canonicalZn₃–ßMT structure. The copper-containing ßMTclusters formed inside the cells were very stable. In contrast,the nascent ß peptide, although it showed cadmium bindingability, produced a highly unstable species, whose stoichiometrydepended upon culture conditions. The absence of ßMT proteinin E.coli protease-proficient hosts grown in cadmium-supplementedmedium pointed to drastic proteolysis of a poorly folded ßpeptide, somehow enhanced by the presence of cadmium. Possiblefunctional and evolutionary implications of the bioactivityof mammalian ßMT in the presence of monovalent and divalentmetal ions are discussed.     

Distinctive properties of signal sequences from bacterial lipoproteins

We have compared a number of attributes (hydrophobicity, aminoacid size, charge and secondary structure propensities) of signalsequences from bacterial lipoproteins with the same attributesof signal peptides from other prokaryotic proteins (non-lipoproteins).Lipoprotein leader sequences tend to be shorter, more hydrophobicand bulky, and they have stronger conformational preferences,the most conspicuous being a predicted ß-turn comprisingpositions 2 or 3 of the mature protein. Another distinctivefeature is a maximum in the local energy profile between positions–1 and +2. With one exception (ß-lactamase III),the lipoproteins do not have Pro in their signal peptides, andthey tend to have fewer Ser and Thr but more Gly than non-lipoproteins.Lipoproteins also lack a net negative charge in the N-terminalregions of the mature proteins. The signal peptides of the bacteriocinplasmid-coded lysis proteins appear to be unique in that theyhave all the ascribed features of lipoprotein signals; thesecharacteristics can be used to guide signal peptide mutagenesisexperiments and to construct new secretion vehicles.     

Synthesis, purification and initial structural characterization of octarellin, a de novo polypeptide modelled on the {alpha}/{beta}-barrel Proteins

We have attempted to construct an artificial polypeptide thatfolds like the eight-stranded parallel ß-barrel structures.Our approach consists of repeating eight times a unit peptidedesigned to adopt a ‘ß-strand/-helix’pattern. A first ‘test’ sequence for this structuralunit was deduced from a series of parameters defined after ananalysis of three natural /ß-barrel proteins and includingprincipally the lengths of the secondary structure elements,the /ß packing and the fitting on average Garnierprofiles. The gene encoding this structural unit was synthesized,cloned and expressed in Escherichia coli either as a monomeror as direct repeats of 2–12 units. Preliminary structuralcharacterization of the 7-, 8- and 9-fold unit polypeptidesby circular dichroism measurements indicates the presence ofthe predicted amount of -helix in the three proteins. Furtheranalysis by urea-gradient gel electrophoresis demonstrates that,in the conditions tested, only the 8-fold unit polypeptide formsa compact structure through a cooperative and rapid two-statefolding transition involving long-range molecular interactions.