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.     

Direct expression in E.coli of a functionally active protein A-snake toxin fusion protein

We constructed a recombinant expression plasmid encoding a proteinA–neurotoxin fusion protein. The fused toxin is directlyexpressed in the periplasmic space of Escherichia coli and canbe purified in the milligram range by a single immuno-affinitystep. The LD₅₀ values of the fused toxin and native toxin are130 and 20 nmol/kg mouse respectively. The K_d values characterizingtheir binding to the nicotinic acetylcholine receptor (AcChoR)are respectively 4.8 ± 0.8 and 0.07 ± 0.03 nM.In contrast, the fused and native toxins are equally well recognizedby a toxin-specific monoclonal antibody which recognizes theAcChoR binding site. The lower toxicity of the fused toxin mightresult, therefore, from a steric hindrance, due to the presenceof the bulky protein A moiety (mol. wt = 31 kd) rather thanto a direct alteration of the ‘toxic’ site. Thefused toxin is more immunogenic than native toxin, since 1 nmolof hybrid toxin and 14 nmol of native toxin give rise to comparabletiters of antitoxin antibodies which, furthermore, are equallypotent at neutralizing neurotoxicity. The work described inthis paper shows that the use of fused toxins may be of paramountimportance for future development of serotherapy against envenomationby snake bites.     

Efficient production of the C-terminal domain of secretory leukoprotease inhibitor as a thrombin-cleavable fusion protein in Escherichia coli

We have developed a high-level production system for the C-terminaldomain of secretory leukoprotease inhibitor (SLPI) to investigateits pharmacological activities. A gene for the C-terminal domainof SLPI, (Asn55-AlalO7)SLPI, was constructed from chemicallysynthesized deoxyoligonucleotides. It was fused to a gene forthe N-terminal portion of human growth hormone via a DNA sequenceencoding Leu-Val-Pro-Arg, which can he cleaved by thrombin.The fused gene was expressed in Escherichia coli under the controlof a trp promoter, and the fusion protein was obtained as aninclusion body. After sulfonation of the cysteine residues,the sulfonated fusion protein was cleaved at the desired siteby thrombin. Sulfonated (Asn55-Ala107)SLPI was refolded in Trisbuffer containing reduced and oxidized glutathione. The resulting(Asn55-Ala107) SLPI was purified by cation-exchange chromatographyand reverse-phase high performance liquid chromatography. Thefinal yield was 50 mg/l culture. (Asn55-Ala107)SLPI was as activeagainst elastase as, but had less trypsin inhibitory activitythan, native SLPI. This system is suitable for the large-scaleproduction of the C-terminal domain of SLPI, which is an elastase-specificinhibitor.     

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.     

Recombinant hirudin: kinetic mechanism for the inhibition of human thrombin

Recombinant hirudin variant-2(Lys⁴⁷ ), was found to be a competitiveinhibitor of human -thrombin with respect to peptidyl p-Miitroanilidesubstrates. These results contrast with those of Degryse andcoworkers that suggest that recombinant hirudin variant-2(Lys⁴⁷)inhibited thrombin by a non-competitive mechanism [Degryse etal. (1989) Protein Engng, 2, 459–465], -Thrombin, whichcan arise from -thrombin by autolysis, was shown to have anaffinity for recombinant hirudin variant-2(Lys⁴⁷) that was fourorders of magnitude lower than that of -thrombin. It was demonstratedthat the apparent noncompetitive mechanism observed previouslywas probably caused by a contamination of the thrombin preparationby -thrombin. Comparison of the inhibition of -thrombin by recombinanthirudins variant-2(Lys⁴⁷) and variant-1, which differ from oneanother in eight out of 65 amino acids, indicated that the twovariants have essentially the same kinetic parameters.     

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     

Cellulose-binding domains: potential for purification of complex proteins

The endoglucanase CenA and the exoglucanase Cex from Cellulomonasfimi each contain a discrete cellulose-binding domain (CBD),at the amino-terminus or carboxyl-terminus respectively. Thegene fragment encoding the CBD can be fused to the gene of aprotein of interest. Using this approach hybrid proteins canbe engineered which bind reversibly to cellulose and exhibitthe biological activity of the protein partner. Alkaline phosphatase(PhoA) from Escherichia coli, and a ß-glucosidase(Abg) from an Agrobacterium sp. are dimeric proteins. The fusionpolypeptides CenA-PhoA and Abg-CBC_cex are sensitive to proteolysisat the junctions between the fusion partners. Proteolysis resultsin a mixture of homo- and heterodimers; these bind to celluloseif one or both of the monomers carry a CBD, e.g. CenA-PhoA/CenA-PhoAand CenA-PhoA/PhoA. CBD fusion polypeptides could be used inthis way to purify polypeptides which associate with the fusionpartner.     

Comparison of conservation within and between the Ser/Thr and Tyr protein kinase family: proposed model for the catalytic domain of the epidermal growth factor receptor

The protein kinase family can be subdivided into two main groupsbased on their ability to phosphorylate Ser/Thr or Tyr substrates.In order to understand the basis of this functional difference,we have carried out a comparative analysis of sequence conservationwithin and between the Ser/Thr and Tyr protein kinases. A multiplesequence alignment of 86 protein kinase sequences was generated.For each position in the alignment we have computed the conservationof residue type in the Ser/Thr, in the Tyr and in both of thekinase subfamilies. To understand the structural and/or functionalbasis for the conservation, we have mapped these conservationproperties onto the backbone of the recently determined structureof the cAMP–dependent Ser/Thr kinase. The results showthat the kinase structure can be roughly segregated, based uponconservation, into three zones. The inner zone contains residueshighly conserved in all the kinase family and describes thehydrophobic core of the enzyme together with residues essentialfor substrate and ATP binding and catalysis. The outer zonecontains residues highly variable in all kinases and representsthe solvent–exposed surface of the protein. The thirdzone is comprised of residues conserved in either the Ser/Thror Tyr kinases or in both, but which are not conserved betweenthem. These are sandwiched between the hydrophobic core andthe solvent-exposed surface. In addition to analyzing overallconservation hi the kinase family, we have also looked at conservationof its substrate and ATP binding sites. The ATP site is highlyconserved throughout the kinases, whereas the substrate bindingsite is more variable. The active site contains several positionswhich differ between the Ser/Thr and Tyr kinases and may beresponsible for discriminating between hydroxyl bearing sidechains. Using this information we propose a model for Tyr substratebinding to the catalytic domain of the epidermal growth factorreceptor (EGFR).     

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.     

Efficient expression and characterization of isolated structural domains of yeast phosphoglycerate kinase generated by site-directed mutagenesis

The two domains of yeast phosphoglycerate kinase were producedby recombinant techniques. The N-domain was obtained by theintroduction of a termination codon at the position coding forPhe185, and the C-domain by a deletion in the gene of the codingsequence between Serl and Leu186. Both domains were efficientlyexpressed in yeast, the level for the C-domain being greaterthan that for the N-domain. Both domains were found to havea quasi-native structure; the C-domain retained its abilityto bind nucleotides. Small local differences were detected indomain structure compared to that in the whole enzyme, probablydue to the lack of interdomain stabilizing interactions. Nevertheless,such an approach provides direct evidence for independent foldingof domains in a two-domain protein.     

Demonstration by burst-phase analysis of a robust folding intermediate in the FF domain

The role of intermediates in the folding reaction of single-domainproteins is a controversial issue. It was previously shown bydifferent methods that an on-pathway intermediate is populatedin the presence of sodium sulphate during the folding of theFF domain from HYPA/FBP11. Here we demonstrate using analysisof the amplitudes of kinetic traces that this burst-phase foldingintermediate is present at different salt concentration andat various pH, and is also found in roughly 30 site-directedmutants. The intermediate appears robust to changing conditionsand thus fulfils an important criterion for a productive molecularspecies on the folding reaction pathway.     

The folding pathway of an engineered circularly permuted PDZ domain

To understand the role of sequence connectivity in the folding pathway of a multi-state protein, we have analysed the folding kinetics of an engineered circularly permuted PDZ domain. This variant has been designed with the specific aim of posing two of the strands participating in the stabilisation of an early folding nucleus as contiguous elements in the primary structure. Folding of the circularly permuted PDZ2 has been explored by a variety of different experimental approaches including stopped-flow and continuous-flow kinetics, as well as ligand-induced folding experiments. Data reveal that although circular permutation introduces a significant destabilisation of the native state, a folding intermediate is stabilised and accumulated prior folding. Furthermore, quantitative analysis of the observed kinetics indicates an acceleration of the early folding events by more than two orders of magnitude. The results support the importance of sequence connectivity both in the mechanism and the speed of protein folding.     

Expression of the amino terminal part of synthetic human growth hormone gene and somatomedin-like activity of expressed protein

We have constructed three different plasmids containing partsof the human growth hormone gene using chemically synthesizedoligomers and cloned them for the purpose of expressing themin Escherichia coli. AB, B and BC gene segments correspondingto ABhGH (residue 1–138), BhGH (residue 44–138)and BChGH (residue 44–192) were placed under the controlof a tryptophan promoter in the expression vector. Upon inductionwith 3-indolylacrylic acid, ABhGH accumulated in cells but theBhGH and BChGH segments were not detected appreciably. Northernblotting analysis showed that the amount of mRNA transcribedfrom the AB gene segment was about ten-fold higher than thatfrom the B or BC gene segment. ABhGH was found to have insulin-likegrowth factor I (IGF-I) activity, which could be explained bythe hydrophilicity curves of these proteins.     

Rapid detection of antigen binding by antibody fragments expressed in the periplasm of Escherichia coli

Bacterial expression systems can greatly facilitate proteinengineering of antibodies. We have developed a system for high-levelexpression of antibodies, antibody fragments, or hybrid antibodieswith novel effector functions in the periplasm of Escherichiacoli. From 5 ml of cells, a simple extraction yields sufficientmaterial for SDS-gel electro-phoresis, detection and characterizationof hapten binding. To demonstrate our system, heavy-chain variableregions and 1 light chains of a mouse anti-NP antibody weresynthesized as hybrid proteins with a bacterial signal peptide(Omp F). Each chain is secreted into the periplasm where processing(cleavage of the signal peptide), folding and heterodimer associationtake place. Periplasmic proteins are released by cold osmoticshock, and hapten-binding activity is easily detected withoutfurther manipulation. The ease of genetic engineering in thissystem will facilitate the production of immunoglobulin derivativesdesigned for specific applications, and expression of thesemolecules in a native state will allow the rapid screening ofcombinatorial libraries and the results of mutagenesis.     

Structural interpretation of site-directed mutagenesis and specificity of the catalytic subunit of protein kinase CK2 using comparative modelling

The catalytic subunit of protein kinase casein kinase 2 (CK2),which has specificity for both ATP and GTP, shows significantamino acid sequence similarity to the cyclin-dependent kinase2 (CDK2). We constructed site-directed mutants of CK2 and useda three-dimensional model to investigate the basis for the dualspecificity. Introduction of Phe and Gly at positions 50 and51, in order to restore the pattern of the glycine-rich motif,did not seriously affect the specificity for ATP or GTP. Weshow that the dual specificity probably originates from theloop situated around the position His115 to Asp120 (HVNNTD).The insertion of a residue in this loop in CK2 subunits, comparedwith CDK2 and other kinases, might orient the backbone to interactwith the base A and G; this insertion is conserved in all knownCK2. The mutant N118, the design of which was based on the modelling,showed reduced affinity for GTP as predicted from the model.Other mutants were intended to probe the integrity of the catalyticloop, alter the polarity of a buried residue and explore theimportance of the carboxy terminus. Introduction of Arg to replaceAsn189, which is mapped on the activation loop, results in amutant with decreased k_cat, possibly as a result of disruptionof the interaction between this residue and basic residues inthe vicinity. Truncation at position 331 eliminates the last60 residues of the subunit and this mutant has a reduced catalyticefficiency compared with the wild-type. Catalytic efficiencyis restored in the truncation mutant by the replacement of apotentially buried Glu at position 252 by Lys, probably owingto a higher stability resulting from the formation of a saltbridge between Lys252 and Asp208.     

Construction and characterization of a single polypeptide chain containing two enzymatically active dihydrofolate reductase domains

A single polypeptide chain containing two dihydrofolate reductase( D M ) sequences from Escherichia coli was constructed to determineif a repeat sequence fusion protein could be expressed in anactive form. The possibility that intersequence interactionscould play a significant role for this enzyme is suggested bythe results of Hall and Frieden (1989, Proc. NatlAcad. Sri.USA, 86, 3060-3064) who observed a substantial decrease in theyield of active enzyme when folded hi the presence of a largeC-terminal fragment. The fusion protein [DHFR(Cysl52Ghi)-De-DHFR(MetlGln)] was efficiently expressed in E.coli cells and hasan activity which is twice that of the wild-type enzyme in thestandard assay. The Michaelis constants of the fusion proteinfor the substrate, dihydrofolate and the cofactor, NADPH, areessentially unchanged from those of the wild-type protein. Theureainduced in vitro unfolding reaction of the fusion proteinat low concentrations was found to be fully reversible and followa three state model, suggesting that the two domains unfoldindependently. At higher protein concentrations the unfoldingtransition broadened and shifted to a higher urea concentration.Size-exclusion chromatography results are consistent with theformation of aggregates at the higher protein concentration,even in the absence of denaturant.     

The location of inhibitory specificities in human mucus proteinase inhibitor (MPI): separate expression of the COOH-terminal domain yields an active inhibitor of three different proteinases

Human mucus proteinase inhibitor (MPI) consists of 107 aminoacids arranged in two domains showing high homology to eachother. This protein is an inhibitor of different serine proteinasesincluding trypsin, chymotrypsin, leukocyte elastase and cathepsinG. On the basis of sequence comparisons it has been suggestedthat the first domain inhibits trypsin, whereas the second onewas thought to be active against chymotrypsin and elastase.To prove the location of the different inhibitory activitiesgene fragments for both domains have been cloned separatelyand expressed in Escherichia coli. Inhibition assays with theisolated recombinant domains showed that the second domain isactive against chymotrypsin, neutrophil elastase and trypsin,whereas for the first domain only a weak activity against trypsincould be detected. These results suggest that the inhibitoryactivities of the native molecule towards these three proteinasesare all located in the second domain.     

Cloning, sequencing and expression of the Fab fragment of a monoclonal antibody to the herbicide atrazine

The Fab region of an IgG2b antibody (AM7B2.1) reactive to theherbicide atrazine was cloned into a plasmid vector using thepolymerase chain reaction and two sets of degenerate oligonucleotideprimers designed to mimic the amino acid variation at the N-terminiof _L-chains and TH-chains. These primers also provide a secretionsignal fused precisely to the antibody gene sequence for secretionof the mature antibody. A further set of universal oligonucleotideprimers was developed for the direct sequencing of the V_H andC_m regions of _B-chains and the V_L and C_L regions of _L-chainswithout subcloning and were used to determine the sequence ofthis antibody. The _L-chain was found to not possess a conservedCys residue at position 23 and the implications of this observationare discussed. The cloned genes were expressed in Escherichiacoli using a commercially available T7 RNA polymerase-basedplasmid. The clones were also expressed in a 17 RNA polymerasebasedsystem containing an attenuated version of the T7 RNA polymerasepromoter, plus a lac promoter placed in an antisense orientation,to enhance plasmid stability. The expressed products were confirmedas atrazine reactive by binding to an atrazine derivative conjugatedwith alkaline phosphatase.