A protease-resistant 61-residue prion peptide causes neurodegeneration in transgenic mice.

An abridged prion protein (PrP) molecule of 106 amino acids, designated PrP106, is capable of forming infectious miniprions in transgenic mice (S. Supattapone, P. Bosque, T. Muramoto, H. Wille, C. Aagaard, D. Peretz, H.-O. B. Nguyen, C. Heinrich, M. Torchia, J. Safar, F. E. Cohen, S. J. DeArmond, S. B. Prusiner, and M. Scott, Cell 96:869-878, 1999). We removed additional sequences from PrP106 and identified a 61-residue peptide, designated PrP61, that spontaneously adopted a protease-resistant conformation in neuroblastoma cells. Synthetic PrP61 bearing a carboxy-terminal lipid moiety polymerized into protease-resistant, beta-sheet-enriched amyloid fibrils at a physiological salt concentration. Transgenic mice expressing low levels of PrP61 died spontaneously with ataxia. Neuropathological examination revealed accumulation of protease-resistant PrP61 within neuronal dendrites and cell bodies, apparently causing apoptosis. PrP61 may be a useful model for deciphering the mechanism by which PrP molecules acquire protease resistance and become neurotoxic.

Structural changes in a hydrophobic domain of the prion protein induced by hydration and by ala-->Val and pro-->Leu substitutions.

X-ray diffraction was used to study the structure of assemblies formed by synthetic peptide fragments of the prion protein (PrP) that include the hydrophobic domain implicated in the Gerstmann-Sträussler-Scheinker (GSS) mutation (P102L). The effects of hydration on polypeptide assembly and of Ala-->Val substitutions in the hydrophobic domain were characterized. Synthetic peptides included: (i) Syrian hamster (SHa) hydrophobic core, SHa106-122 (KTNMKHMAGAAAAGAVV); (ii) SHa104-122(3A-V), with A-->V mutations at 113, 115 and 118 (KPKTNMKHMVGVAAVGAVV); (iii) mouse (Mo) wild-type sequence of the N-terminal hydrophobic domain, Mo89-143WT; and (iv) the same mouse sequence with leucine substitution for proline at residue number 101, Mo89-143(P101L). Samples of SHa106-122 that formed assemblies while drying under ambient conditions showed X-ray patterns indicative of 33 A thick slab-like structures having extensive H-bonding and intersheet stacking. By contrast, lyophilized peptide that was equilibrated against 100 % relative humidity showed assemblies with only a few layers of beta-sheets. The Ala-->Val substitutions in SHa104-122 and Mo89-143(P101L) resulted in the formation of 40 A wide, cross-beta fibrils. Observation of similar size beta-sheet fibrils formed by peptides SHa104-122(3A-V) and the longer Mo89-143(P101L) supports the notion that the hydrophobic sequence forms a template or core that promotes the beta-folding of the longer peptide. The substitution of amino acids in the mutants, e.g. 3A-->V and P101L, enhances the folding of the peptide into compact structural units, significantly enhancing the formation of the extensive beta-sheet fibrils.

Binding of neural cell adhesion molecules (N-CAMs) to the cellular prion protein.

To identify molecular interaction partners of the cellular prion protein (PrP(C)), we sought to apply an in situ crosslinking method that maintains the microenvironment of PrP(C). Mild formaldehyde crosslinking of mouse neuroblastoma cells (N2a) that are susceptible to prion infection revealed the presence of PrP(C) in high molecular mass (HMM) protein complexes of 200 to 225 kDa. LC/MS/MS analysis identified three murine splice-variants of the neural cell adhesion molecule (N-CAM) in the complexes, which isolate with caveolae-like domains (CLDs). Enzymatic removal of N-linked sugar moieties did not disrupt the complexes, arguing that the interaction of PrP with N-CAM occurs through amino acid side-chains. Additionally, similar levels of PrP/N-CAM complexes were found in N2a and prion-infected N2a (ScN2a) cells. With the use of an N-CAM-specific peptide library, the PrP-binding site was determined to comprise beta-strands C and C' within the two consecutive fibronectin type III (FNIII) modules found in proximity of the membrane-attachment site of N-CAM. As revealed by in situ crosslinking of PrP deletion mutants, the PrP face of the binding site is formed by the N terminus, helix A (residues 144-154) and the adjacent loop region of PrP. N-CAM-deficient (N-CAM(-/-)) mice that were intracerebrally challenged with scrapie prions succumbed to disease with a mean incubation period of 122 (+/-4.1, SEM) days, arguing that N-CAM is not involved in PrP(Sc) replication. Our findings raise the possibility that N-CAM may join with PrP(C) in carrying out some as yet unidentified physiologic cellular function.

A synthetic peptide initiates Gerstmann-Sträussler-Scheinker (GSS) disease in transgenic mice.

The molecular basis of the infectious, inherited and sporadic forms of prion diseases is best explained by a conformationally dimorphic protein that can exist in distinct normal and disease-causing isoforms. We identified a 55-residue peptide of a mutant prion protein that can be refolded into at least two distinct conformations. When inoculated intracerebrally into the appropriate transgenic mouse host, 20 of 20 mice receiving the beta-form of this peptide developed signs of central nervous system dysfunction at approximately 360 days, with neurohistologic changes that are pathognomonic of Gerstmann-Sträussler-Scheinker disease. By contrast, eight of eight mice receiving a non-beta-form of the peptide failed to develop any neuropathologic changes more than 600 days after the peptide injections. We conclude that a chemically synthesized peptide refolded into the appropriate conformation can accelerate or possibly initiate prion disease.

Copper binding to octarepeat peptides of the prion protein monitored by mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) was used to measure the binding of Cu2+ ions to synthetic peptides corresponding to sections of the sequence of the mature prion protein (PrP). ESI-MS demonstrates that Cu2+ is unique among divalent metal ions in binding to PrP and defines the location of the major Cu2+ binding site as the octarepeat region in the N-terminal domain, containing multiple copies of the repeat ProHisGlyGlyGlyTrpGlyGln. The stoichiometries of the complexes measured directly by ESI-MS are pH dependent: a peptide containing four octarepeats chelates two Cu2+ ions at pH 6 but four at pH 7.4. At the higher pH, the binding of multiple Cu2+ ions occurs with a high degree of cooperativity for peptides C-terminally extended to incorporate a fifth histidine. Dissociation constants for each Cu2+ ion binding to the octarepeat peptides, reported here for the first time, are mostly in the low micromolar range; for the addition of the third and fourth Cu2+ ions to the extended peptides at pH 7.4, K(D)'s are <100 nM. N-terminal acetylation of the peptides caused some reduction in the stoichiometry of binding at both pH's. Cu2+ also binds to a peptide corresponding to the extreme N-terminus of PrP that precedes the octarepeats, arguing that this region of the sequence may also make a contribution to the Cu2+ complexation. Although the structure of the four-octarepeat peptide is not affected by pH changes in the absence of Cu2+, as judged by circular dichroism, Cu2+ binding induces a modest change at pH 6 and a major structural perturbation at pH 7.4. It is possible that PrP functions as a Cu2+ transporter by binding Cu2+ ions from the extracellular medium under physiologic conditions and then releasing some or all of this metal upon exposure to acidic pH in endosomes or secondary lysosomes.

Abnormal births and other "ill omens" : The adaptive case for infanticide.

We summarize the ethnographic literature illustrating that "abnormal birth" circumstances and "ill omens" operate as cues to terminate parental investment. A review of the medical literature provides evidence to support our assertion that ill omens serve as markers of biological conditions that will threaten the survival of infants. Daly and Wilson (1984) tested the prediction that children of demonstrably poor phenotypic quality will be common victims of infanticide. We take this hypothesis one stage further and argue that some children will be poor vehicles for parental investment yet are not of demonstrably poor quality at birth. We conclude that when people dispose of infants due to "superstitious beliefs" they are pursuing an adaptive strategy in eliminating infants who are poor vehicles for parental investment.

Sleeping like a baby: attitudes and experiences of bedsharing in northeast England.

This paper reports findings from a study that investigated infant care practices in a small population of Northeast England in order to determine whether parent-infant bedsharing is common parenting behavior. In a year-long prospective study we examined the opinions and practices of parents with regard to their infants' nighttime sleeping strategies before and after the birth of their babies. Results confirm that parents pursue a heterogeneous array of nighttime parenting strategies and that 65 percent of the sample had actually bedshared. Parents with no previous intention to do so slept with their babies for a variety of reasons. One of this study's most important findings is that babies were being brought into bed with both parents. Ninety five percent of the bedsharing infants slept with both mother and father. This study has shown that bedsharing is a relatively common parenting practice. Despite initial worries and fears, mainly concerning overlaying, some parents found bedsharing an effective option yet were covert in their practices, fearing the disapproval of health professionals and relatives.

Randomised trial of infant sleep location on the postnatal ward.

OBJECTIVE: To determine whether postnatal mother-infant sleep proximity affects breastfeeding initiation and infant safety. DESIGN: Randomised non-blinded trial analysed by intention to treat. SETTING: Postnatal wards of the Royal Victoria Hospital (RVI), Newcastle upon Tyne, UK. PARTICIPANTS: 64 newly delivered mother-infant dyads with a prenatal intention to breastfeed (vaginal deliveries, no intramuscular or intravenous opiate analgesics taken in the preceding 24 h). INTERVENTION: Infants were randomly allocated to one of three sleep conditions: baby in mother's bed with cot-side; baby in side-car crib attached to mother's bed; and baby in stand-alone cot adjacent to mother's bed. MAIN OUTCOME MEASURES: Breastfeeding frequency and infant safety observed via night-time video recordings. RESULTS: During standardised 4-h observation periods, bed and side-car crib infants breastfed more frequently than stand-alone cot infants (mean difference (95% confidence interval (CI)): bed v stand-alone cot = 2.56 (0.72 to 4.41); side-car crib v stand-alone cot = 2.52 (0.87 to 4.17); bed v side-car crib = 0.04 (-2.10 to 2.18)). No infant experienced adverse events; however, bed infants were more frequently considered to be in potentially adverse situations (mean difference (95% CI): bed v stand-alone cot = 0.13 (0.03 to 0.23); side-car crib v stand-alone cot = 0.04 (-0.03 to 0.12); bed v side-car crib = 0.09 (-0.03-0.21)). No differences were observed in duration of maternal or infant sleep, frequency or duration of assistance provided by staff, or maternal rating of postnatal satisfaction. CONCLUSION: Suckling frequency in the early postpartum period is a well-known predictor of successful breastfeeding initiation. Newborn babies sleeping in close proximity to their mothers (bedding-in) facilitates frequent feeding in comparison with rooming-in. None of the three sleep conditions was associated with adverse events, although infrequent, potential risks may have occurred in the bed group. Side-car cribs are effective in enhancing breastfeeding initiation and preserving infant safety in the postnatal ward.

Triadic bed-sharing and infant temperature.

The effects on infants of sleeping with their parents is currently the subject of much debate. One concern regarding infants who sleep in their parents' bed involves the possibility of overheating. Previous research reported a significantly greater core temperature of 0.1 degrees C among a cohort of bed-sharing infants compared with a matched cohort of infants sleeping alone. This paper presents a preliminary analysis of the overnight rectal temperature of 12 of the 20 infants who were monitored sleeping alone and with their parents on separate nights at the University of Durham Parent-Infant Sleep Lab. No significant differences were found in all night rectal temperature, or temperature from 2 h after sleep onset between bed-sharing and cot sleeping nights. These preliminary analyses suggest a night-time difference in rectal temperature between routine bed-sharers and routine cot sleepers, however, these findings will be further explored in the full analyses for this study.

Purification of synthetic peptides using reversible chromatographic probes based on the Fmoc molecule.

A rapid, versatile, reversible procedure for purifying synthetic peptides has been developed based on the specific incorporation of 4-carboxylate Fmoc derivatives onto the terminal amino acid of peptidyl-resins. The acid stable 4-COR-Fmoc derivatives were synthesised with a variety of chemical groups thus altering the chromatographic properties of the "target" peptides and permitting their convenient purification, either by reversed-phase HPLC or ion exchange chromatography. The assembly of the peptides involved a capping step to prevent the formation of deletion forms. The 4-COR-Fmoc derivatives were incorporated either as preformed amino acid conjugates or as activated succinimidyl esters. After HF cleavage and purification the 4-COR-Fmoc probes were quantitatively removed with organic bases. The efficiency of the technique was demonstrated by the purification of small to large sized peptides, including a cyclic analogue.

The prevalence and characteristics associated with parent-infant bed-sharing in England.

AIMS: To investigate the characteristics of parent-infant bed-sharing prevalence in England. METHODS: Data on night-time sleeping practices from a two year, local, longitudinal study and a three-year, national, cross-sectional study were obtained. A total of 261 infants in North Tees were followed up at 1 and 3 months of age, as were 1095 infants aged 1 week to 1 year from five English health regions. RESULTS: Data from both studies found that almost half of all neonates bed-shared at some time with their parents (local = 47%, 95% CI 41 to 54; national = 46%, 95% CI 34 to 58), and on any one night in the first month over a quarter of parents slept with their baby (local = 27%, 95% CI 22 to 33; national = 30%, 95% CI 20 to 42). Bed-sharing was not related to younger mothers, single mothers, or larger families, and was not more common in the colder months, at weekends, or among the more socially deprived families; in fact bed-sharing was more common among the least deprived in the first months of life. Breast feeding was strongly associated with bed-sharing, both at birth and at 3 months. Bed-sharing prevalence was uniform with infant age from 3 to 12 months; on any one night over a fifth of parents (national = 21%, 95% CI 18 to 24) slept with their infants. CONCLUSION: Bed-sharing is a relatively common practice in England, not specific to class, but strongly related to breast feeding.

Application of reversible biotinylated label for directed immobilization of synthetic peptides and proteins: isolation of ligates from crude cell lysates.

Chaperonin 10 protein from Rattus norvegicus (Rat cpn 10) has been reported to bind chaperonin 60 from Escherichia coli (GroEL) in an ATP-dependent manner. Chemically synthesized Rat cpn10 was immobilized in a defined orientation to agarose-bound monomeric avidin using a reversible biotinylated affinity label (1), attached to the N alpha-terminal residue. The resulting affinity chromatographic matrix was then used to isolate binding proteins from a crude cell lysate. Following affinity separation the bound ligand and ligate was released by treatment with organic base. Rat cpn 10 was prepared using a highly effective synthetic protocol involving HBTU/HOBt activation and capping with N-(2-chlorobenzyloxycarbonyloxy) succinimide to terminate unreacted amino groups. The biotinylated Fmoc-based molecule (1) was introduced specifically onto the N alpha-terminal amino acid as the succinimidyl carbonate, before final cleavage and deprotection of side-chain protecting groups using a low-TFMSA/high-HF procedure. Crude biotinylated Rat cpn10 (Rat cpn10 + 1) was immobilized on monomeric avidin with a binding efficiency of approximately 75% and unlabelled truncated/capped impurities eluted off the column with buffer. The biotinylated Rat cpn10-avidin affinity matrix was then used to isolate GroEL from a crude cell lysate. The identity of the purified protein was confirmed by SDS-PAGE and binding to a specific anti-GroEL monoclonal antibody (MoAb). These results extend the applicability of the biotinylated label (1), providing a reversible non-covalent anchor for immobilization of peptide and protein ligands, thus simplifying isolation of ligates and enabling recovery of synthetic material under mild conditions.

Insurance ovulation, embryo mortality and twinning.

The 'insurance ova' hypothesis (Anderson, 1990) views dizygotic twinning as a by-product of selection for multiple ovulation which sometimes--in error--results in the birth of twins. From this viewpoint, polyovulation is a mechanism which reduces the risks of fertilization failure or embryo defect/mortality. If DZ twin births are a 'side-effect' of a mechanism which compensates for defective embryos one might predict that embryo defect rates and twinning rates will covary. This prediction is tested using national-level data on twinning rates and rates of trisomy-21 (Down's syndrome), and a strong positive correlation is found, even when controlling for maternal age. One suggestion that follows from this finding is that intra- and interpopulation variation in both twinning rates and Down's syndrome rates may result, in part, from individual variation in pre-implantation rejection of embryos in the very early stages of pregnancy. In this paper it is proposed that the 'insurance ova' explanation for twinning in humans could be expanded to incorporate a model of rejection of anomalous embryos, be they anomalies of number or type. Variation in the efficiency of an embryo rejection mechanism, combined with variation in frequency of polyovulation, would have consequences for individual reproductive success.

Chemical synthesis and purification of proteins: a methodology.

Classical stepwise solid-phase peptide synthesis (SPPS) has been used successfully for the synthesis of proteins up to 150 residues in length, although usually with poor yields and homogeneity. The major limitation has been the inability to separate chromatographically similar deletion and truncated impurities from the target sequence. We have developed a highly effective protocol for stepwise SPPS and 'one-step' purification of small proteins. to demonstrate the effectiveness of the methodology we synthesised the 101 residue chaperonin 10 protein from Rattus norvegicus (Rat Cpn 10) using three different chemical protocols. Highly homogeneous Rat Cpn10 was obtained using an optimised synthetic strategy and one-step purification procedure (method C), involving (i) HBTU/HOBt activation, (ii) N-(2-chlorobenzyloxycarbonyloxy)succinimide as capping agent and (iii) the incorporation of a reversible Fmoc-based chromatographic probe, derivatised with a lipophilic group for fast one-step RP purification, to give an overall yield of 9.6%. Analysis by ESI-MS indicated that the product was virtually free of deletion impurities, while RP-HPLC under four different conditions and CZE indicated that the protein was 100 and 84% pure, respectively. The spontaneous folding of Rat Cpn10 into its biologically active form was found to correlate well with the degree of purity as assessed by chromatography, ESI-MS and sequencing, since 29 (A), 55 (B) and 81% (C) of correctly folded heptameric structure was obtained. The degree of homogeneity was also reflected in the ability of purified Rat Cpn10 to facilitate the refolding of yeast enolase.

Affinity purification of 101 residue rat cpn10 using a reversible biotinylated probe.

The purification of large synthetic peptides using conventional separation techniques often results in poor yields and homogeneity due to the accumulation of chromatographically similar deletion and truncated impurities. We have developed a highly effective synthetic strategy and one-step purification procedure that is based on (i) the application of single coupling using HBTU/HOBt activation to reduce incomplete couplings, (ii) the use of N-(2-chlorobenzyloxycarbonyloxy)succinimide as a capping agent to terminate deletion sequences and (iii) the N-terminal derivatization of the complete peptidyl-resin with a reversible Fmoc-based chromatographic probe possessing enhanced physico-chemical properties (i.e. hydrophobicity, charge or affinity label). We report the application of a biotinylated probe, activated as the succinimidyl carbonate, for the purification of a 101 residue chaperonin protein from Rattus norvegicus (rat cpn10), previously synthesized using an optimized synthetic protocol. Biotinylated rat cpn10 was separated from underivatized impurities on an immobilized monomeric avidin column. Free rat cpn10 was released from avidin-agarose column with 5% aqueous triethylamine and after desalting by RP-HPLC gave 9.9% recovery. Characterization and assessment of homogeneity was achieved using ESI-MS, CZE and RP-HPLC.

Mycobacterium tuberculosis chaperonin 10 and N-truncated fragments. Their synthesis and purification by the isoelectric focusing technique carried out in solution.

The Mycobacterium tuberculosis chaperonin 10 protein and fragments corresponding to sequences 59-99, 51-99 and 26-99 were synthesised by the solid-phase methodology using a double coupling protocol and without the aid of capping agents. After the final acid cleavage using the low TFMSA-high HF protocol the polypeptides were purified by either the ion exchange chromatography/RP-HPLC combination or the isoelectric separation carried out in solution and followed by semi-preparative RP-HPLC. Comparison of the results obtained through the two approaches indicated that in general the isoelectricfocusing/HPLC combination was superior both in terms of recovery of final material and its purity. The advantages found were as follows: (i) Unlike ion exchange chromatography, no tailoring of the separation conditions is required, (ii) Several consecutive focusings can be carried out in progressively narrower pH gradients. This increases the separation resolution without the need of changing other separation parameters, (iii) Very little manipulation is needed, and each focusing requires 3-5h. (iv) Full compatibility with non-ionic denaturants such as 8 M urea. This increases solubility so that using the ROTOFOR instrument described here 50-100 mg crude polypeptide can be processed daily. Thus the isoelectric focusing technique carried out in solution is a valid and inexpensive alternative to ion exchange chromatography.

The use of crown ethers in peptide chemistry-V. Solid-phase synthesis of peptides by the fragment condensation approach using crown ethers as non-covalent protecting groups.

We have previously described the conditions by which peptide synthesis by the solid-phase fragment condensation approach can be carried out using crown ethers as non-covalent protection for the N alpha-amino group. Here we demonstrate that the procedure can be extended to large, partially protected peptide fragments possessing free Lys and/or Arg residues. The first step was to ensure that complex formation on the side chain of amino acids was not detrimental to the methodology and exhibited the same solubility and coupling properties as N alpha-complexed peptides. Thus, a model hexapeptide was synthesized using Fmoc chemistry containing Lys and Arg residues, which, when complexed with 18-Crown-6, was readily soluble in DCM and coupled quantitatively to a resin-bound tetrapeptide. Two tripeptides were then prepared, one containing a free Ser residue, the other free Tyr, to examine the possible occurrence of side reactions. After coupling using standard conditions only the former tripeptide exhibited the formation of the O-acylation by-product (5%). Another model hexapeptide containing Lys, Tyr, Ser and Asp protected with a TFA-stable adamantyl group was complexed with 18-Crown-6 and coupled to the resin-bound tetrapeptide with near quantitative yield. Extending the length of the peptide to 21 and 40 residues, which represent sequences Gly52 to Leu72 (21-mer) and Pro33 to Leu72 (40-mer) from Rattus norvegicus chaperonin 10 protein, respectively, resulted in partially protected fragments that were readily soluble in water, thus enabling purification by RP-HPLC. Complexation with 18-Crown-6 gave two highly soluble products that coupled to resin-board tetramer with 68% and 50% coupling efficiencies for the 21-mer and 40-mer, respectively. Treatment with 1% DIEA solutions followed by acidolytic cleavage and purification of the major product confirmed that the correct product has been formed, when analysed by amino acid analysis and ESI-MS. These results served to extend the methodology of non-covalent protection of large partially protected peptide fragments for the stepwise fragment condensation of polypeptides.

The structural and aggregation properties of the synthetic C-terminal half (104-mer) polypeptide from HIV p24gag resemble those of full-length protein.

The aggregation and structural properties of the synthetic C-terminal half [Ala330, Ala350(270-373; 104-mer)] polypeptide from HIV-1 p24gag were studied. In concentrated solutions the synthetic polypeptide aggregated to tetramers which, upon dilution, gave a mixture of monomeric and dimeric and dimeric species. These results correlated well with the in vitro aggregation properties of recombinant p24. The tetrameric form of the synthetic polypeptide had a pI which differed by about four units from that of the mixture of monomeric and dimeric species. CD studies indicated that the latter contained, in aqueous solutions, a compact molecule lacking, however, a defined tertiary structure. Addition of MeOH to aqueous solutions of both tetramer and monomer/dimer mixture induced a more defined structure, which was assigned to that of an alpha + beta protein in agreement with secondary structure predictions. A model of the dimeric form of the 104-mer, which takes into account the results presented here and those from a study on the specificity of a set of anti-104-mer MoAbs, is presented. Finally, the results indicated that the structure of the 104-mer in its dimeric form is similar to that adopted by the same sequence when part of full-length p24.

The interaction of myristylated peptides with the catalytic domain of protein kinase C revealed by their sequence palindromy and the identification of a myristyl binding site.

Using a model of the enzyme structure and the results from a series of free and myristylated peptides, we provide evidence that peptides corresponding to the pseudosubstrate sequence of protein kinase C bind to the enzyme substrate binding site in an essentially extended conformation. This and the nearly symmetrical location of positive charges around the substrate phosphoritable site allow the peptide to bind to the enzyme in either an N-to-C orientation or its C-to-N opposite orientation. The latter is favoured by a change in residue chirality or when the peptide bears a myristoyl chain at its N-terminus. A myristyl binding site was also identified in the enzyme structure and its location in a region proximal to the C-terminal residue of pseudosubstrate bound in the N-to-C direction suggested that C-myristylation of peptide substrates should be more effective than N-myristoylation in antagonizing the enzyme. A peptide (H-RFARKGALRQKN-CONH-Myr) which contains the myristyl chain covalently linked to the C-terminal residue of the pseudosubstrate was thus made and shown to be a potent inhibitor of the histone kinase reaction of protein kinase C and the phosphorylation of p47 in intact cells.

Chemical synthesis and characterisation of rat chaperonin 10: effect of chain length, ions, heat and N-terminal acetylation on unchaperoned folding into its heptameric form.

Recently, the sequence of mitochondrial chaperonin 10 from Rattus norvegicus (rat cpn10), with N-terminal acetylation, has been published. Two syntheses of rat cpn10 were performed, the first using a classical carbodiimide-mediated double coupling protocol (Method A) and the second a more efficient HBTU/HOBT/single coupling procedure (Method B). The latter also involved the application of a capping procedure, using N-(2-chlorobenzyloxycarbonyloxy)succinimide [Z(2-Cl)-OSu]. The crude protein from Method A was purified using a two-step isoelectric focusing/RP-HPLC scheme and found to contain a high proportion of a deletion peptide (less Gln60). Conversely, rat cpn10 from Method B was purified to homogeneity by one-step RP-HPLC, using a reversible lipophilic chromatographic probe. The proportion of biologically active heptameric structure was directly related to the purity of the protein and attained 84% with material from Method B. The addition of Ca/Mg ions, pH 7.2, or a heating/cooling cycle increased the proportion of heptamer for less pure protein. Shorter sequences were found not to fold into heptamers, suggesting that aggregation/folding motifs are located in 1-25 and 77-101 regions of rat cpn10. The heptameric cpn10 (Method B) bound correctly to GroEL from E. coli, demonstrating that N-terminal acetylation is not necessary for its folding and binding to bacterial cpn60.