Chimeric ribonuclease as a source of human adapter protein for targeted drug delivery

Assembled modular complexes for targeted drug delivery can bebased on strong non-covalent interactions between a cargo modulecontaining an adapter protein and a docking tag fused to a targetingprotein. We have recently constructed a completely humanizedadapter/docking tag system based on interactions between 15amino acid (Hu-tag) and 110 amino acid (HuS) fragments of humanribonuclease I (RNase I). Although recombinant HuS can be expressedand refolded into a functionally active form, the purificationprocedure is cumbersome and expensive, and more importantly,it yields a significant proportion of improperly folded proteins.Here we describe engineering, high-yield expression, and purificationof a chimeric bovine/human RNase (BH-RNase) comprising 1–29N-terminal amino acids of bovine ribonuclease A and 30–127amino acids of human RNase I. Unlike RNase I, the chimeric BH-RNasecan be cleaved by either subtilisin or proteinase K betweenA20 and S21, providing a functionally active HuS. The HuS obtainedfrom chimeric BH-RNase differs from wild-type HuS by an N24Tsubstitution; therefore, we have reverted this substitutionby mutating N24 to T24 in BH-RNase. This BH-RNase mutant canalso be cleaved by subtilisin or proteinase K yielding wild-typeHuS. The affinity of HuS obtained from BH-RNase to Hu-tag isapproximately five times higher than that for recombinant HuS,reflecting a higher percentage of properly folded proteins. Received June 9, 2003; revised August 4, 2003; accepted August 28, 2003.     

Improved antigenicity of the HIV env protein by cleavage site removal

The HTV env glycoprotein mediates virus infection and cell fusionthrough an interaction with the CD4 molecule present at thesurface of T4⁺ lymphocytes. Although env presents a major antigenictarget, vaccinia recombinants expressing env elicit low titresof anti-env antibody (Kieny et at., Bio/Technology, 4, 790–795,1986). To delimit the functional domains of env and to improvethe immunogenicity of the vaccinia recombinants we constructedvariants expressing env proteins in which the site permittingcleavage of the gp160 precursor to yield gp120 and gp41 wasremoved, the gp120 and gp41 moieties separated or in which thesignal sequence and hydrophobic domains were replaced by equivalentsfrom rabies virus G. Analysis of variants revealed that thegp120 moiety is alone capable of interacting with CD4 and ofprovoking aggregation of T4⁺ lymphocytes, whereas cell-associatedgp41 liberated by gp160 cleavage was essential for cell fusion.The identity of the signal and transmembrane zones however appearedunimportant. Although removal of the consensus sequence permittingcleavage of gp160 prevented syncytium formation but not aggregationof T4⁺ lymphocytes, significant cleavage continued to take place.Removal of a second potential cleavage site blocked gp160 cleavage.The live viruses were examined for immunogenicity: recombinant1139 which lacks both putative cleavage sites was found to elicita 10-fold higher antibody response in experimental animals thanthe parental recombinant. Received May 17, 1988; accepted June 10, 1988.     

Overproduction of bovine {beta}-casein in Escherichia coli and engineering of its main chymosin cleavage site

A cDNA clone containing the entire coding region for bovineß-casein A³ flanked by 53 base pairs of 5' non-codingand 358 base pairs of 3' non-coding sequences was isolated froma bovine mammary cDNA phagemid library. The coding segment formature ß-casein was subcloned into the T7 expressionsystem, in which the expression of recombinant ß-caseinwas controlled by the T7 gene 10 promoter and ribosome bindingsite. High level expression of Met-ß-casein to 20%of the total soluble proteins was obtained in Escherichia coliwithin 2 h after induction of T7 RNA-polymerase synthesis. Inan attempt to induce secretion the coding segment for matureß-casein was coupled to the ompA translations initiationsignal and signal peptide coding sequence but no secretion ofthe fusion protein and no processing of the signal peptide fromthe fusion protein was observed. Instead, the Met-ß-caseincould be isolated in asoluble form from E.coli cells after anosmotic shock, indicative of a periplasmic location. This proceduredid not lyse the cells. The protein was purified to homogeneityafter a pH 4.8 isoelectric precipitation followed by reversed-phasehigh-performance liquid chromatography. The ß-caseincDNA was altered to change the main chymosin cleavage siteinß-casein at position 192–193 in two ways, namelyfrom Leu–Tyr to Pro–Pro and to Leu–stop. Thesemutations were designed to prevent generation of the bitterpeptide ßcasein(193–209) by chymosin cleavage.The mutant Met-ß-caseins were expressed in E.colito the same level as wild-type Met-ß-casein. Purifiedmutant Met-ß-casein(Prol92– Prol93) was no longerhydrolysed by chymosin at the 192–193 bond.     

Prediction of protein secondary structure with a reliability score estimated by local sequence clustering

Most algorithms for protein secondary structure prediction arebased on machine learning techniques, e.g. neural networks.Good architectures and learning methods have improved the performancecontinuously. The introduction of profile methods, e.g. PSI-BLAST,has been a major breakthrough in increasing the prediction accuracyto close to 80%. In this paper, a brute-force algorithm is proposedand the reliability of each prediction is estimated by a z-scorebased on local sequence clustering. This algorithm is intendedto perform well for those secondary structures in a proteinwhose formation is mainly dominated by the neighboring sequencesand short-range interactions. A reliability z-score has beendefined to estimate the goodness of a putative cluster foundfor a query sequence in a database. The database for predictionwas constructed by experimentally determined, non-redundantprotein structures with <25% sequence homology, a list maintainedby PDBSELECT. Our test results have shown that this new algorithm,belonging to what is known as nearest neighbor methods, performedvery well within the expectation of previous methods and thatthe reliability z-score as defined was correlated with the reliabilityof prediction. This led to the possibility of making very accuratepredictions for a few selected residues in a protein with anaccuracy measure of Q₃ > 80%. The further development ofthis algorithm, and a nucleation mechanism for protein foldingare suggested. Received March 27, 2003; revised June 30, 2003; accepted August 22, 2003.     

Structure-based substitutions for increased solubility of a designed protein

Manipulation of protein solubility is important for many aspectsof protein design and engineering. Previously, we designed aseries of consensus ankyrin repeat proteins containing one,two, three and four identical repeats (1ANK, 2ANK, 3ANK and4ANK). These proteins, particularly 4ANK, are intended for useas a universal scaffold on which specific binding sites canbe constructed. Despite being well folded and extremely stable,4ANK is soluble only under acidic conditions. Designing interactionswith naturally occurring proteins requires the designed proteinto be soluble at physiological pH. Substitution of six leucineswith arginine on exposed hydrophobic patches on the surfaceof 4ANK resulted in increased solubility over a large pH range.Study of the pH dependence of stability demonstrated that 4ANKis one of the most stable ankyrin repeat proteins known. Inaddition, analogous leucine to arginine substitutions on thesurface of 2ANK allowed the partially folded protein to assumea fully folded conformation. Our studies indicate that replacementof surface-exposed hydrophobic residues with positively chargedresidues can significantly improve protein solubility at physiologicalpH. Received June 23, 2003; revised August 22, 2003; accepted August 28, 2003.     

Role of tryptophan 121 in the soluble CuA domain of cytochrome c oxidase: structure and electron transfer studies

To investigate the contribution of tryptophan-121 (Trp121) residueto the structure and function of soluble Cu_A domain of cytochromec oxidase, three mutant proteins, Trp121Tyr, Trp121Leu and Trp121-deletedmutant of the soluble domain of Paracoccus versutus cytochromec oxidase, were constructed and expressed in Escherichia coliBL21 (DE3). Optical spectral studies showed that both the coordinationstructure of the Cu_A center and the secondary structure of theprotein were changed significantly in the Leu substitution anddeletion mutants of Trp121. Their electron transfer activitywith cytochrome c was inhibited severely, as shown in stopped-flowkinetic studies. However, the Cu_A center can be reconstructedin the Trp121Tyr mutant although its stability decreases comparedwith the wild-type protein. This mutant keeps the same secondarystructure as the wild-type protein, but can only transfer electronswith cytochrome c at a rate of one-seventh-fold. Based on theinformation on the structure, we also investigated and analyzedthe possible factors that affect electron transfer. It appearsthat the aromatic ring, the size of the side chain and the hydrogenbonding ability of the Trp121 are crucial to the structure andfunction of the soluble Cu_A domain. Received September 11, 2002; revised February 26, 2003; accepted May 27, 2003.     

Immobilized oxidoreductase as an additive for refolding inclusion bodies: application to antibody fragments

Three foldases—the apical domain of GroEL (mini-chaperone)and two oxidoreductases (DsbA and DsbC) from Escherichia coli—werestudied in refolding a protein with immunoglobulin fold (immunoglobulin-foldedprotein) that had been produced as inclusion bodies in E.coli.The foldases promoted the refolding of single-chain antibodyfragments from denaturant-solubilized and reduced inclusionbodies in vitro, and also effectively functioned as alternativesfor labilizing agent and oxidizing reagent in the stepwise dialysissystem. Immobilization of the oxidoreductases enhanced refoldingand recovery of functional single-chain antibody in the dialysissystem, suggesting that immobilized oxidoreductases can be usedas an effective additive for refolding immunoglobulin-foldedproteins in vitro. Received April 7, 2003; revised June 5, 2003; accepted June 6, 2003.     

Effect of mutations involving charged residues on the stability of staphylococcal nuclease: a continuum electrostatics study

A continuum electrostatics model is used to calculate the relativestabilities of 117 mutants of staphylococcal nuclease (SNase)involving the mutation of a charged residue to an unchargedresidue. The calculations are based on the crystallographicstructure of the wild-type protein and attempt to take implicitlyinto account the effect of the mutations in the denatured stateby assuming a linear relationship between the free energy changescaused by the mutation in the native and denatured states. Agood correlation (linear correlation coefficient of 0.8) isfound with published experimental relative stabilities of thesemutants. The results suggest that in the case of SNase (i) chargedresidues contribute to the stability of the native state mainlythrough electrostatic interactions, and (ii) native-like electrostaticinteractions may persist in the denatured state. The continuumelectrostatics method is only moderately sensitive to modelparameters and leads to quasi-predictive results for the relativemutant stabilities (error of 2–3 kJ mol^–1 or ofthe order of k_BT), except for mutants in which a charged residueis mutated to glycine. Received March 24, 2003; revised August 11, 2003; accepted September 12, 2003.     

Structure-oriented rational design of chymotrypsin inhibitor models

Three peptides modelling a highly potent, 35-residue chymotrypsininhibitor (Schistocerca gregaria chymotrypsin inhibitor) weredesigned and synthesized by convergent peptide synthesis. Foreach model peptide, the inhibitory constant (K_i) on chymotrypsinand the solution structure were determined. In addition, moleculardynamics calculations were performed for all of them. Two modelscontaining approximately half of the parent inhibitor (17 of35 residues) were designed and subsequently found to have nosubstantial inhibitory activity (K_i values in the mM range).The third model composed of 24 amino acid residues proved tobe an effective (K_i 10^–7) inhibitor of bovine chymotrypsin.Both the solution structure properties determined by NMR spectroscopyand the dynamic behaviour of the latter model system are comparableto the native inhibitor. In contrast, the structure and dynamicsof the first two related model peptides show characteristicdifferences. We suggest that the conformation and flexibilityof the modelled protease inhibitor are crucial for its biologicalefficiency. Moreover, the structural and dynamic features ofthe binding loop (28–33) and those of the rest of themolecule appear to be interdependent. Most importantly, thesestructural characteristics can be rationally modified, at leastpartially, by peptide design. Received March 7, 2003; revised August 25, 2003; accepted August 26, 2003.     

A systematic approach for yielding a potential pool of enzymes: practical case for chiral resolution of (R,S)-ketoprofen ethyl ester

A systematic approach for the selection of potential biocatalystsfrom a natural source was developed and then a practical applicationwas addressed. The approach that involves systematically combinedconventional screening methods and current tools comprises thefollowing consecutive steps: strain enrichment for activityscreening, identification of positive strains, choosing wholegenome-sequenced strains as candidates, gathering informationabout responsible enzymes, bioinformatic analyses and gene mining,probing genetic molecules and then functional expression. Thetarget compound (R,S)-ketoprofen ethyl ester was to be resolvedinto an enantiomer, and a potential esterase from Pseudomonasfluorescens KCTC 1767 was prepared by the proposed procedure.The enzyme had a high activity and also strict selectivity forthe enantiomer (S)-ketoprofen and was suitable therefore asa biocatalyst for practical use. The result achieved by thecombined approach could not easily be obtained using other approacheswith typical procedures. Hence the approach proposed here shouldbe of considerable use for the screening of potential enzymes,particularly for enzymes with desired activity to unnaturalsubstrates, from conditionally expressed and/or repressed proteinsthat are distributed widely in natural pools under normal conditions. Received July 4, 2002; revised January 20, 2003; accepted April 4, 2003.     

Optimizing the search algorithm for protein engineering by directed evolution

An in silico protein model based on the Kauffman NK-landscape,where N is the number of variable positions in a protein andK is the degree of coupling between variable positions, wasused to compare alternative search strategies for directed evolution.A simple genetic algorithm (GA) was used to model the performanceof a standard DNA shuffling protocol. The search effectivenessof the GA was compared to that of a statistical approach calledthe protein sequence activity relationship (ProSAR) algorithm,which consists of two steps: model building and library design.A number of parameters were investigated and found to be importantfor the comparison, including the value of K, the screeningsize, the system noise and the number of replicates. The statisticalmodel was found to accurately predict the measured activitiesfor small values of the coupling between amino acids, K 1.The ProSAR strategy was found to perform well for small to moderatevalues of coupling, 0 K 3, and was found to be robust to noise.Some implications for protein engineering are discussed. Received January 2, 2003; revised May 13, 2003; accepted June 19, 2003.     

PROSPECT II: protein structure prediction program for genome-scale applications

A new method for fold recognition is developed and added tothe general protein structure prediction package PROSPECT (http://compbio.ornl.gov/PROSPECT/).The new method (PROSPECT II) has four key features. (i) We havedeveloped an efficient way to utilize the evolutionary informationfor evaluating the threading potentials including singletonand pairwise energies. (ii) We have developed a two-stage threadingstrategy: (a) threading using dynamic programming without consideringthe pairwise energy and (b) fold recognition considering allthe energy terms, including the pairwise energy calculated fromthe dynamic programming threading alignments. (iii) We havedeveloped a combined z-score scheme for fold recognition, whichtakes into consideration the z-scores of each energy term. (iv)Based on the z-scores, we have developed a confidence index,which measures the reliability of a prediction and a possiblestructure–function relationship based on a statisticalanalysis of a large data set consisting of threadings of 600query proteins against the entire FSSP templates. Tests on severalbenchmark sets indicate that the evolutionary information andother new features of PROSPECT II greatly improve the alignmentaccuracy. We also demonstrate that the performance of PROSPECTII on fold recognition is significantly better than any othermethod available at all levels of similarity. Improvement inthe sensitivity of the fold recognition, especially at the superfamilyand fold levels, makes PROSPECT II a reliable and fully automatedprotein structure and function prediction program for genome-scaleapplications. Received March 20, 2003; revised June 28, 2003; accepted July 8, 2003.     

Stable, soluble T-cell receptor molecules for crystallization and therapeutics

Antibody and T-cell receptors (TCRs) are the primary recognitionmolecules of the adaptive immune system. Antibodies have beenextensively characterized and are being developed for a largenumber of therapeutic applications. This has been possible becauseof the ability to manufacture stable, soluble, monoclonal antibodieswhich retain the antigen specificity of B cells. Unlike antibodies,TCRs are not expressed in a soluble form, but are anchored tothe T-cell surface by an insoluble trans-membrane domain. Characterizationand development of TCRs has been hampered by the lack of suitablemethods for producing them as soluble and stable proteins. Herewe report the engineering of soluble human TCRs suitable forcrystallization studies and potentially for in vivo therapeuticuse. Received March 11, 2003; revised July 1, 2003; accepted July 30, 2003.     

A long insertion reverts the functional effect of a substitution in acetylcholinesterase

Proteins are thought to undertake single substitutions, deletionsand insertions to explore the fitness landscape. Nevertheless,the ways in which these different kind of mutations act togetherto alter a protein phenotype remain poorly described. We introducedincrementally the single substitution W290A and a 26 amino acidlong insertion at the 297 location in the Nippostrongylus brasiliensisacetylcholinesterase B sequence and analysed in vitro the inducedchanges in the hydrolysis rate of three hemi-substrates: pirimicarb,paraoxon methyl and omethoate. The substitution decreased thehydrolysis rate of the three hemi-substrates. The insertiondid not influence this kinetic alteration induced by the substitutionfor the former hemi-substrate, but reverted it for the two others.These results show that two different kinds of mutations caninteract together to influence the direction of a protein’sadaptative walk on the fitness landscape. Received January 28, 2003; revised April 25, 2003; accepted June 6, 2003.     

Inhibition of the CD28-CD80 co-stimulation signal by a CD28-binding affibody ligand developed by combinatorial protein engineering

CD28 is one of the key molecules for co-stimulatory signallingin T cells. Here, novel ligands (affibodies) showing selectivebinding to human CD28 (hCD28) have been selected by phage displaytechnology from a protein library constructed through combinatorialmutagenesis of a 58-residue three-helix bundle domain derivedfrom staphylococcal protein A. Analysis of selected affibodiesshowed a marked sequence homology and biosensor analyses showedthat all investigated affibodies bound to hCD28 with micromolaraffinities (K_D). No cross-reactivity towards the related proteinhuman CTLA-4 could be observed. This lack of cross-reactivityto hCTLA-4 suggests that the recognition site on hCD28 for theaffibodies resides outside the conserved MYPPPYY motif. Theapparent binding affinity for hCD28 could be improved throughfusion to an Fc fragment fusion partner, resulting in a divalentpresentation of the affibody ligand. For the majority of selectedanti-CD28 affibodies, in co-culture experiments involving JurkatT-cells and CHO cell lines transfected to express human CD80(hCD80) or LFA-3 (hLFA-3) on the cell surface, respectively,pre-incubation of Jurkat cells with affibodies resulted in inhibitionof IL-2 production when they were co-cultured with CHO (hCD80⁺)cells, but not with CHO (hLFA-3⁺) cells. For one affibody variantdenoted Z_CD28:5 a clear concentration-dependent inhibition wasseen, indicating that this affibody binds hCD28 and specificallyinterferes in the interaction between hCD28 and hCD80. Received March 11, 2003; revised June 30, 2003; accepted July 30, 2003.     

Synthesis of a novel histidine analogue and its efficient incorporation into a protein in vivo

Proteins containing unnatural amino acids have immense potentialin biotechnology and medicine. We prepared several histidineanalogues including a novel histidine analogue, ß-(1,2,3-triazol-4-yl)-DL-alanine.These histidine analogues were assayed for translational activityin histidine-auxotrophic Escherichia coli strain UTH780. Weobserved that several histidine analogues, including our novelhistidine analogue, were efficiently incorporated into the proteinin vivo; however, other analogues were rejected. These resultssuggest that the hydrogen atom at a specific position seriouslyaffects incorporation. Received April 10, 2003; revised June 20, 2003; accepted July 22, 2003.     

Vicinal disulfide turns

The formation of a disulfide bond between adjacent cysteineresidues is accompanied by the formation of a tight turn ofthe protein backbone. In nearly 90% of the structures analyzeda type VIII turn was found. The peptide bond between the twocysteines is in a distorted trans conformation, the omega torsionangle ranges from 159 to –133°, with an average valueof 171°. The constrained nature of the vicinal disulfideturn and the pronounced difference observed between the oxidizedand reduced states, suggests that vicinal disulfides may beemployed as a ‘redox-activated’ conformational switch. Received December 16, 2002; revised June 30, 2003; accepted July 30, 2003.     

Expression of the catalytic subunit of cAMP-dependent protein kinase in Escherichia coli: multiple isozymes reflect different phosphorylation states

The catalytic subunit of mouse cAMP-dependent protein kinaseexpressed in Escherichia coliwas separated into three distinctspecies using Mono-S ion exchange chromatography. These isozymescorresponded to three isodectric variants with pIs of 6.4 (30%),7.2 (60%) and 8.2 (10%). The Stokes' radius of each form was27.7, 27.1 and 26.3 Å respectively. Using electrospraymass spectroscopy the differences between the isozymes wereshown to be due to phosphorylation, with each form differingby 80 mass units corresponding to a single phosphate. The fullyphosphorylated recombinant enzyme contained four phosphateswhile the dominant isozymecontained only three. Since the enzymeis not phosphorylated when active site mutations are introducedinto the C-subunit, these phosphates are incorporated in anautocatalytic mechanism and are not due to E.coli protein kinases.When the recombinant enzyme was compared with the mammalianporcine heart enzyme significant differences in posttranslationalmodifications were observed. The mammalian enzyme could alsobe separated into two isozymes. However, in contrast to therecombinant enzyme, the mammalian isozymes displayed an identicalmass of 40 840. This correlated with two different post-translationalmodifications: two phosphates and an N-terminal myristyl moiety.The importance of post-translational modifications, and inparticularthe phosphorylation state, for the expression of eukaryoticproteins in E.coli is discussed. Received March 25, 1993; revised May 25, 1993; accepted June 3, 1993.     

Change in the crystal packing of soybean {beta}-amylase mutants substituted at a few surface amino acid residues

In spite of the high similarity of amino acid sequence and three-dimensionalstructure between soybean ß-amylase (SBA) and sweetpotato ß-amylase (SPB), their quaternary structureis quite different, being a monomer in SBA and a tetramer inSPB. Because most of the differences in amino acid sequencesare found in the surface region, we tested the tetramerizationof SBA by examining mutations of residues located at the surface.We designed the SBA tetramer using the SPB tetramer structureas a model and calculating the change of accessible surfacearea (ASA) for each residue in order to select sites for themutation. Two different mutant genes encoding SB3 (D374Y/L481R/P487D)and SB4 (K462S added to SB3), were constructed for expressionin Escherichia coli and the recombinant proteins were purified.They existed as a monomer in solution, but gave completely differentcrystals from the native SBA. The asymmetric unit of the mutantscontains four molecules, while that of native SBA contains one.The interactions of the created interfaces revealed that therewere more intermolecular interactions in the SB3 than in theSB4 tetramer. The substituted charged residues on the surfaceare involved in interactions with adjacent molecules in a differentway, forming a new crystal packing pattern. Received June 9, 2003; revised September 11, 2003; accepted September 16, 2003.