Peptide Affinity Column Research Articles

Biomimetic affinity chromatography for antibody purification: Host cell protein binding and impurity removal

Peptide affinity chromatography has received increasing attention as an alternative to protein A chromatography in antibody purification. However, its lower selectivity than protein A chromatography has impeded its success in practical applications. In particular, efficient removal of contaminants, including host cell proteins (HCPs) and DNA, is a great challenge for peptide affinity chromatography in monoclonal antibody (mAb) manufacturing. In this work, a biomimetic peptide ligand (bPL), FYWHCLDE, was coupled onto Sepharose 6 Fast Flow (SepFF) to synthesize a peptide affinity gel, SepFF-bPL, for the investigation of the binding mechanism of HCP as well as the feasibility of antibody capture. The results showed that the SepFF-bPL column exhibited effective removal of mAb aggregates as well as mAb capture from feedstocks of various origins, whereas poor removal of HCP and DNA was found. Mechanistic studies of HCP binding indicated that electrostatic interactions dominated HCP binding on the SepFF-bPL gel and that ionic conductivity had a significant influence on HCP binding at low salt concentrations. Thus, combined chromatin extraction and anion exchange adsorption were introduced prior to SepFF-bPL chromatography for initial contaminant removal to reduce mAb aggregation induced by HCP and the loading burden of contaminants in SepFF-bPL chromatography. A proof-of-concept study of the purification train demonstrated a high recovery of mAb (68.7%) and low levels of HCP (23 ppm) and DNA (below the limit of detection) in the final product, which were acceptable for the mandatory requirements in clinical applications. This research provided a deep understanding of HCP binding on the peptide affinity column and led to the development of an effective purification train.

Peptide Affinity Purification of Antibodies.

This protocol describes antibody purification using a peptide affinity column. Peptides can be designed that use naturally occurring cysteines within the protein target's primary sequence, or a cysteine can be added to either end of the peptide to provide free thiols for attachment. The peptides can then be covalently attached to resins bearing thiol-reactive linkers. The most commonly used thiol-reactive moieties are iodoacetyl and maleimide, both of which react selectively with peptides containing cysteine thiols. Although gravity can be used to cycle the antibody solution (e.g., serum) over the column (it is recommended that the antibody be cycled multiple times to obtain maximal yield), the use of a pump to apply the serum to the column in a continuous flow manner improves the yield of antibody. Similarly, washing the column after application of the antibody without and with 0.5 m NaCl should be performed with at least 20 column volumes.

Affinity chromatography of human IgG with octapeptide ligands identified from eleven peptide-ligand candidates

We have reported earlier on the characterization of four peptides from a library of 15 peptide ligand candidates for human IgG (hIgG) obtained by a biomimetic design strategy and identified three high-affinity octapeptides. In this work, the left 11 peptides were evaluated and we found two more octapeptides (FYCHWQDE and FYCHNQDE) that showed high affinity for hIgG. The binding pH ranges for the two ligands were different, but the optimum pH values were the same for each other (pH 6.0). Both the ligands showed high specificity and bound hIgG mainly by electrostatic interactions. Ligand binding competition experiments revealed that the binding sites on hIgG for the two octapeptides were similar to those for Protein A. Finally, hIgG was purified from human serum with high purities and recovery yields with the two peptide affinity columns. Thus, among the 15 peptide candidates, a total of five octapeptides were identified as high-affinity ligands of hIgG. The five ligands were all derived from the same peptide model FYxHxxxE (where x denotes any amino acid) and contained four common hot spots F132, Y133, H137, and E143 of the affinity motif of Protein A. Analyses and evaluation of the peptide library would help deepen our understanding of the affinity binding of Protein A to IgG, and promote application of the biomimetic strategy in the design of affinity ligands for different proteins.

AB0136 Clinical Aspects and Pathogenicity of Anti-Cyclic Citrullinated Glucose-6-Phospate Isomerase Peptide (CCG) Antibodies in Patients with Rheumatoid Arthritis

BackgroundAnti-citrullinated protein antibodies (Abs) (ACPA) have been reported as a hallmark of diagnosis and disease progression in sera of patients with rheumatoid arthritis (RA). Although several ACPA targeting citrullinated fibrinogen,...

Phosphatidylinositol 3-Kinase (PI3K) Activity Bound to Insulin-like Growth Factor-I (IGF-I) Receptor, which Is Continuously Sustained by IGF-I Stimulation, Is Required for IGF-I-induced Cell Proliferation

Continuous stimulation of cells with insulin-like growth factors (IGFs) in G(1) phase is a well established requirement for IGF-induced cell proliferation; however, the molecular components of this prolonged signaling pathway that is essential for cell cycle progression from G(1) to S phase are unclear. IGF-I activates IGF-I receptor (IGF-IR) tyrosine kinase, followed by phosphorylation of substrates such as insulin receptor substrates (IRS) leading to binding of signaling molecules containing SH2 domains, including phosphatidylinositol 3-kinase (PI3K) to IRS and activation of the downstream signaling pathways. In this study, we found prolonged (>9 h) association of PI3K with IGF-IR induced by IGF-I stimulation. PI3K activity was present in this complex in thyrocytes and fibroblasts, although tyrosine phosphorylation of IRS was not yet evident after 9 h of IGF-I stimulation. IGF-I withdrawal in mid-G(1) phase impaired the association of PI3K with IGF-IR and suppressed DNA synthesis the same as when PI3K inhibitor was added. Furthermore, we demonstrated that Tyr(1316)-X-X-Met of IGF-IR functioned as a PI3K binding sequence when this tyrosine is phosphorylated. We then analyzed IGF signaling and proliferation of IGF-IR(-/-) fibroblasts expressing exogenous mutant IGF-IR in which Tyr(1316) was substituted with Phe (Y1316F). In these cells, IGF-I stimulation induced tyrosine phosphorylation of IGF-IR and IRS-1/2, but mutated IGF-IR failed to bind PI3K and to induce maximal phosphorylation of GSK3β and cell proliferation in response to IGF-I. Based on these results, we concluded that PI3K activity bound to IGF-IR, which is continuously sustained by IGF-I stimulation, is required for IGF-I-induced cell proliferation.

Dermatopontin Promotes Epidermal Keratinocyte Adhesion via α3β1 Integrin and a Proteoglycan Receptor

Dermatopontin, an extracellular matrix component initially purified from bovine dermis, promoted cell adhesion of the human epidermal keratinocyte cell line (HaCaT cells). HaCaT cells spread on dermatopontin and formed actin fibers. Adhesion of HaCaT cells to dermatopontin was inhibited by both EDTA and heparin and was mediated in part by alpha3beta1 integrin. A synthetic peptide (DP-4, PHGQVVVAVRS; bovine dermatopontin residues 33-43) specifically inhibited adhesion of cells to dermatopontin, and when the DP-4 peptide was coated on the well, it promoted cell adhesion in a dose-dependent manner. An active core sequence of the DP-4 peptide was localized to an eight-amino acid sequence (GQVVVAVR). These results indicate that dermatopontin is a novel epidermal cell adhesion molecule and suggest that the DP-4 sequence is critical for the cell adhesive activity of dermatopontin. Adhesion of cells to DP-4 was strongly inhibited by heparin. When HaCaT cells were treated with heparitinase I, the cells failed to adhere to DP-4 but chondroitinase ABC treatment did not influence the adhesion activity. DP-4 specifically interacted with biotinylated heparin, and this interaction was inhibited by unlabeled heparin. DP-4 peptide significantly promoted the adhesion of cells overexpressing syndecans, and syndecan bound to a DP-4 peptide affinity column. These results suggest that HaCaT cells adhere to dermatopontin through alpha3beta1 integrin and a heparan sulfate proteoglycan-type receptor, which is likely a syndecan. We conclude that dermatopontin plays a role as a multifunctional adhesion molecule for epidermal cells.

Transcription Factor Epiprofin Is Essential for Tooth Morphogenesis by Regulating Epithelial Cell Fate and Tooth Number

In tooth morphogenesis, the dental epithelium and mesenchyme interact reciprocally for growth and differentiation to form the proper number and shapes of teeth. We previously identified epiprofin (Epfn), a gene preferentially expressed in dental epithelia, differentiated ameloblasts, and certain ectodermal organs. To identify the role of Epfn in tooth development, we created Epfn-deficient mice (Epfn-/-). Epfn-/- mice developed an excess number of teeth, enamel deficiency, defects in cusp and root formation, and abnormal dentin structure. Mutant tooth germs formed multiple dental epithelial buds into the mesenchyme. In Epfn-/- molars, rapid proliferation and differentiation of the inner dental epithelium were inhibited, and the dental epithelium retained the progenitor phenotype. Formation of the enamel knot, a signaling center for cusps, whose cells differentiate from the dental epithelium, was also inhibited. However, multiple premature nonproliferating enamel knot-like structures were formed ectopically. These dental epithelial abnormalities were accompanied by dysregulation of Lef-1, which is required for the normal transition from the bud to cap stage. Transfection of an Epfn vector promoted dental epithelial cell differentiation into ameloblasts and activated promoter activity of the enamel matrix ameloblastin gene. Our results suggest that in Epfn-deficient teeth, ectopic nonproliferating regions likely bud off from the self-renewable dental epithelium, form multiple branches, and eventually develop into supernumerary teeth. Thus, Epfn has multiple functions for cell fate determination of the dental epithelium by regulating both proliferation and differentiation, preventing continuous tooth budding and generation.

A peptide affinity column for the identification of integrin α IIb-binding proteins

To understand the regulation of integrin α IIbβ 3, a critical platelet adhesion molecule, we have developed a peptide affinity chromatography method using the known integrin regulatory motif, LAMWKVGFFKR. Using standard Fmoc chemistry, this peptide was synthesized onto a Toyopearl AF-Amino-650 M resin on a 6-aminohexanoic acid (Ahx) linker. Peptide density was controlled by acetylation of 83% of the Ahx amino groups. Four recombinant human proteins (CIB1, PP1, ICln and RN181), previously identified as binding to this integrin regulatory motif, were specifically retained by the column containing the integrin peptide but not by a column presenting an irrelevant peptide. Hemoglobin, creatine kinase, bovine serum albumin, fibrinogen and α-tubulin failed to bind under the chosen conditions. Immunodetection methods confirmed the binding of endogenous platelet proteins, including CIB1, PP1, ICln RN181, AUP-1 and β 3-integrin, from a detergent-free platelet lysate. Thus, we describe a reproducible method that facilitates the reliable extraction of specific integrin-binding proteins from complex biological matrices. This methodology may enable the sensitive and specific identification of proteins that interact with linear, membrane-proximal peptide motifs such as the integrin regulatory motif LAMWKVGFFKR.

A Structural Basis for the Acute Effects of HIV Protease Inhibitors on GLUT4 Intrinsic Activity

Human immunodeficiency virus (HIV) protease inhibitors (PIs) act as reversible noncompetitive inhibitors of GLUT4 with binding affinities in the low micromolar range and are known to contribute to alterations in glucose homeostasis during treatment of HIV infection. As aspartyl protease inhibitors, these compounds all possess a core peptidomimetic structure together with flanking hydrophobic moieties. To determine the molecular basis for GLUT4 inhibition, a family of related oligopeptides containing structural elements found in PIs was screened for their ability to inhibit 2-deoxyglucose transport in primary rat adipocytes. The peptide oxybenzylcarbonyl-His-Phe-Phe-O-ethyl ester (zHFFe) was identified as a potent inhibitor of zero-trans glucose flux with a K(i) of 26 mum. Similar to PIs, transport inhibition by this peptide was acute, noncompetitive, and reversible. Within a Xenopus oocyte expression system, zHFFe acutely and reversibly inhibited GLUT4-mediated glucose uptake, whereas GLUT1 activity was unaffected at concentrations as high as 1 mm. The related photoactivatable peptide zHFF-p-benzoylphenylalanine-[(125)I]Tyr-O-ethyl ester selectively labeled GLUT4 in rat adipocytes and indinavir effectively protected against photolabeling. Furthermore, GLUT4 bound to a peptide affinity column containing the zHFF sequence and was eluted by indinavir. These data establish a structural basis for PI effects on GLUT4 activity and support the direct binding of PIs to the transport protein as the mechanism for acute inhibition of insulin-stimulated glucose uptake.

Production and Characterization of a New Antibody Specific for the Mutant EGF Receptor, EGFRvIII, in Camelus bactrianus

EGFRvIII is the type III deletion mutant form of the epidermal growth factor receptor (EGFR) with transforming activity. This tumor-specific antigen is ligand independent, contains a constitutively active tyrosine kinase domain and has been shown to be present in a number of human malignancies. In this study, we report the production and characterization of camel antibodies that are directed against the external domain of the EGFRvIII. Antibodies developed in camels are smaller (i.e. IgG2 and IgG3 subclasses lack light chains) than any other conventional mammalian antibodies. This property of camel antibodies makes them ideal tools for basic research and other applications such as tumor imaging and cancer therapy. In the present study, camel antibodies were generated by immunization of camelids (Camelus bactrianus and Camelus dromedarius) with a synthetic 14-amino acid peptide corresponding to the mutated sequence of the EGFR, tissue homogenates of several patients with human glioblastoma, medulloblastoma and aggressive breast carcinoma, as well as EGFR-expressing cell lines. Three subclasses of camel IgG [conventional (IgG1, 160 kD) and heavy chain-only antibodies (IgG2 and IgG3, 90 kD)] were separated by their different binding properties to protein A and protein G affinity columns. The anti-EGFRvIII peptide antibodies from immunized camels were purified further using the EGFRvIII synthetic peptide affinity column. The purified anti-EGFRvIII peptide camel antibodies selectively bound to the EGFRvIII peptide and affinity-purified EGFRvIII from malignant tissues and detected a protein band of 140 kD from malignant tissues by Western blot. Affinity analysis showed that the antibodies from C. bactrianus and C. dromedarius reacted with peptide and antigen purified from a small cell lung cancer ascitic fluid with affinities of 2 × 10⁸ and 5 × 10⁷M^–1 to the same extent, respectively. Since the functional antigen-binding domain of the anti-EGFRvIII antibodies in camels is much simpler and located only on the heavy chains of proteins, we are currently developing recombinant and smaller versions of the variable domain of these naturally occurring heavy-chain antibodies (V_HH) for use in tumor imaging and cancer therapy.

Separation of enzymatically active and inactive prostate-specific antigen (PSA) by peptide affinity chromatography.

Prostate-specific antigen (PSA) is a serine protease with highly prostate-specific expression and an important marker for prostate cancer. We have previously identified novel PSA-binding peptides that enhance the enzymatic activity of PSA when produced as fusion proteins. PSA-binding peptides and derivatives with a spacer were chemically synthesized and used to prepare an affinity column, which was used to fractionate PSA in seminal plasma, serum, and LNCap cell culture medium. Approximately 67% of seminal plasma PSA bound to the peptide affinity column and was eluted under mild conditions. Eluted PSA was intact and enzymatically active while the unbound fraction mainly contained various nicked forms. ProPSA from LNCap cells bound to the peptide column only after activation by trypsin. PSA-binding peptides can be used to separate enzymatically active and inactive forms of PSA. Thus the peptides are potentially useful as ligands for development of methods for specific detection of active free PSA.

Generation of an affinity column for antibody purification by intein-mediated protein ligation

Coupling an antigenic peptide to a solid support is a crucial step in the affinity purification of a peptide-specific antibody. Conventional methods for generating reactive agarose, cellulose or other matrices for peptide conjugation are laborious and can result in a significant amount of chemical waste. In this report, we present a novel method for the facile production of a peptide affinity column by employing intein-mediated protein ligation (IPL) in conjunction with chitin affinity chromatography. A reactive thioester was generated at the C-terminal of the chitin binding domain (CBD) from the chitinase A1 of Bacillus circulans WL-2 by thiol-induced cleavage of the peptide bond between the CBD and a modified intein. Peptide epitopes possessing an N-terminal cysteine were ligated to the chitin bound CBD tag. We demonstrate that the resulting peptide columns permit the highly specific and efficient affinity purification of antibodies from animal sera.

HSP90, HSP70, and GAPDH Directly Interact with the Cytoplasmic Domain of Macrophage Scavenger Receptors

The macrophage scavenger receptor (MSR) is a trimeric membrane protein which binds to modified low-density lipoprotein (LDL) and has been indicated in the development of atherosclerosis. It has recently been demonstrated that the N-terminal cytoplasmic domain of MSR has an important role in the efficient internalization and cell-surface expression of the receptor. This study shows that the N-terminal cytoplasmic domain in bovine was constructed using a peptide architecture technique in which the peptide chain was bundled at their C-terminus to yield a trimeric form and that this did not form an ordered structure. Furthermore, the binding proteins to the cytoplasmic domain of MSR were determined for the first time using a peptide affinity column. Sequence analyses of the specific binding proteins in bovine revealed that heat shock protein 90 (HSP90), heat shock protein 70 (HSP70), leucine aminopeptidase (LAP), adenocylhomocysteinase, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were included. GST-pull-down assay and immunoprecipitation analyses on HSP90, HSP70, and GAPDH showed that all these proteins could bind to the cytoplasmic domain of MSR in vitro and in vivo. These proteins interact with the cytoplasmic domain directly and may have an effect on the functions of MSR such as internalization, cell-surface expression, and signal transduction.

Characterization of Hoxd1 protein-DNA-binding specificity using affinity chromatography and random DNA oligomer selection.

1. Hoxd1 is member of the labial subfamily of Hox genes that has a conserved 60 amino acid homeodomain region. The homeodomain is an important determining factor in the binding of the protein to specific DNA sequence(s). DNA-binding specificity for the Hoxd1 protein has not been determined previously. 2. We have employed a rapid affinity chromatography method to determine optimal DNA binding sequences for the 109 amino acid Hoxd1 peptide, comprising the homeodomain and the entire carboxy terminal region of the Hoxd1 protein. 3. Labial Hox proteins have intrinsically weak DNA-binding activity that has been attributed to the nonbasic residues at positions 2 and 3 in the N-terminal arm of the homeodomain. The presence of the Hoxd1 carboxy terminal region negated the influence of the nonbasic residues and facilitated Hoxd1 DNA-binding specificity. 4. DNA sequences bound to the Hoxd1 peptide-affinity column were separated from a random pool of oligonucleotide sequences by gradient elution and enriched by polymerase chain reaction. Preferred sequences were identified on 5' and 3' of a TAAT core, extending the binding site to T/AT/gTAATTGTA. 5. Stability and specificity of optimal DNA-binding sequence for Hoxd1 homeodomain were determined by equilibrium and kinetic studies. Dissociation coefficient constant (KD) was estimated to be 8.6 x 10(-9) M and the DNA-Hoxd1 homeodomain complex has a half life (t(1/2)) of 12.7 min. 6. A molecular model of Hoxd1 homeodomain-DNA interaction based on the X-ray coordinates of Antennapedia homeodomain-DNA complex has revealed novel interactions of key Hoxd1 residues at the protein-DNA interface.

Rapid identification of key amino-acid–DNA contacts through combinatorial peptide synthesis

Background: Basic helix–loop–helix (bHLH) transcription factors are characterized by a conserved four-helix bundle that recognizes a specific hexanucleotide DNA sequence in the major groove. Previous studies have shown that amino acids in the basic region make base-specific contacts, whereas the HLH region is responsible for dimerization. Structural data suggest that portions of the loop region may be proximal to the DNA; however, the role of the loop in DNA-binding affinity and specificity has not been investigated. Results: Protein–DNA recognition by the Drosophila bHLH transcription factor Deadpan was probed using combinatorial solid-phase peptide synthesis methods. A series of bHLH peptide libraries that modulate amino acid content and length in the loop region was screened with DNA and peptide affinity columns, and analyzed using matrix-assisted laser desorption ionization mass spectrometry (MALDI–MS). A functional bHLH peptide with reduced loop length was found, and Lys80 was unambiguously identified as the sole loop residue critical for DNA binding. Unnatural amino acids were substituted at this position to assess contributions of the terminal amino group and the alkyl chain length to DNA-binding affinity and specificity. Conclusions: Using combinatorial solid-phase peptide synthesis methods and MALDI–MS, we were able to rapidly identify a key amino acid involved in DNA binding by a bHLH protein. Our approach provides a powerful alternative to current recombinant DNA methods to identify and probe the energetics of protein–DNA interactions.

Regulation of c-Myc through Phosphorylation at Ser-62 and Ser-71 by c-Jun N-Terminal Kinase

The expression of c-myc promotes cell proliferation and also sensitizes cells to various extracellular apoptotic stimuli. However, signal pathways regulating the function of Myc proteins during apoptosis are unknown. c-Jun N-terminal kinase (JNK) is activated by various apoptotic stimuli, but neither the target molecule(s) or the action of JNK has been identified in Myc-mediated apoptosis. Here, we found that JNK selectively interacted with, and phosphorylated, c-Myc at Ser-62 and Ser-71 as confirmed with phospho-c-Myc-specific antibodies. Interestingly, dominant negative mutant JNK(APF) impaired the c-Myc-dependent apoptosis, but not mutated c-Myc (S62A/S71A)-dependent apoptosis triggered by UV irradiation. Furthermore, c-Myc (S62A/S71A)-expressing NIH3T3 cells were not sensitized like wild type c-Myc-expressing NIH3T3 cells to JNK-activating apoptotic stimuli, such as UV and Taxol. These results indicate that the JNK pathway is selectively involved in the c-Myc-mediated apoptosis and that the apoptotic function of c-Myc is directly regulated by JNK pathway through phosphorylation at Ser-62 and Ser-71.

Disruption of the p70(s6k)/p85(s6k) gene reveals a small mouse phenotype and a new functional S6 kinase.

Recent studies have shown that the p70(s6k)/p85(s6k) signaling pathway plays a critical role in cell growth by modulating the translation of a family of mRNAs termed 5'TOPs, which encode components of the protein synthetic apparatus. Here we demonstrate that homozygous disruption of the p70(s6k)/p85(s6k) gene does not affect viability or fertility of mice, but that it has a significant effect on animal growth, especially during embryogenesis. Surprisingly, S6 phosphorylation in liver or in fibroblasts from p70(s6k)/p85(s6k)-deficient mice proceeds normally in response to mitogen stimulation. Furthermore, serum-induced S6 phosphorylation and translational up-regulation of 5'TOP mRNAs were equally sensitive to the inhibitory effects of rapamycin in mouse embryo fibroblasts derived from p70(s6k)/p85(s6k)-deficient and wild-type mice. A search of public databases identified a novel p70(s6k)/p85(s6k) homolog which contains the same regulatory motifs and phosphorylation sites known to control kinase activity. This newly identified gene product, termed S6K2, is ubiquitously expressed and displays both mitogen-dependent and rapamycin-sensitive S6 kinase activity. More striking, in p70(s6k)/p85(s6k)-deficient mice, the S6K2 gene is up-regulated in all tissues examined, especially in thymus, a main target of rapamycin action. The finding of a new S6 kinase gene, which can partly compensate for p70(s6k)/p85(s6k) function, underscores the importance of S6K function in cell growth.

Purification of Human α2-Antiplasmin with Chicken IgY Specific to Its Carboxy-Terminal Peptide

ABSTRACT α2-antiplasmin, a plasma glycoprotein of the serpin superfamily, is the primary physiological inhibitor of plasmin, the key enzyme in fibrin degradation. Previous purification methods utilize lengthy multistep protocols with low yields or use monoclonal antibodies that are expensive or difficult to make. With a relatively small investment, a chicken was immunized with keyhole limpet hemocyanin-conjugated to α2-antiplasmin C-terminal 26 residue synthetic peptide and the peptide-specific antibody (IgY) was isolated from the egg yolks of hens using the peptide affinity column. Based on the interaction between this IgY and α2-antiplasmin, pure α2-antiplasmin was isolated from human plasma in two steps: (a) citrated plasma was precipitated with 15% PEG-8000 to remove the bulk of plasma proteins while retaining the majority of α2-antiplasmin activity; and (b) the α2-antiplasmin was affinity-purified from the supernatant using the IgY column. Yields were typically 48% and the purity and authenticity of the α2-antiplasmin were verified by gel electrophoresis, Western Blot analysis, N-terminal sequence, and amino acid analysis.

Production of Antipeptide Antisera

This unit describes methods used for the chemical coupling of synthetic peptides to carrier proteins, required for the preparation of peptide immunogens. The carrier protein described here is keyhole limpet hemocyanin (KLH) because it is the one most commonly used. However, other proteins may be used in place of KLH, including bovine serum albumin (BSA) and ovalbumin. Coupling may be accomplished as described with MBS, which requires a Cys residue in the peptide, or with glutaraldehyde, EDCI, or BDB. Also included are an assay for detecting free sulfhydryl (SH) groups, a means of calculating coupling efficiency, an immunization schedule, an indirect ELISA, and a method for preparing a peptide affinity column. The final methodology described is the multiple antigen (MAP) system.

A function for the QKRAA amino acid motif: mediating binding of DnaJ to DnaK. Implications for the association of rheumatoid arthritis with HLA-DR4.

The amino acid motif QKRAA, when expressed on HLA-DRB1, carries susceptibility to develop rheumatoid arthritis. This motif is the basis of strong B and T cell epitopes. Furthermore, it is highly overrepresented in protein databases, suggesting that it carries a function of its own. To identify this function, we used QKRAA peptide affinity columns to screen total protein extracts from Escherichia coli. We found that DnaK, the E. coli 70-kD heat shock protein, binds QKRAA. Of interest, DnaK has a natural ligand, DnaJ, that contains a QKRAA motif. We found that QKRAA-containing peptides inhibit the binding of DnaK to DnaJ. Furthermore, rabbit antibody to the QKRAA motif can inhibit binding of DnaJ to DnaK. These data suggest that QKRAA mediates the binding of E. coli chaperone DnaJ to its partner chaperone DnaK.