Identification and characterization of a -1 reading frameshift in the heavy chain constant region of an IgG1 recombinant monoclonal antibody produced in CHO cells

Frameshifts lead to complete alteration of the intended amino acid sequences, and therefore may affect the biological activities of protein therapeutics and pose potential immunogenicity risks. We report here the identification and characterization of a novel -1 frameshift variant in a recombinant IgG1 therapeutic monoclonal antibody (mAb) produced in Chinese hamster ovary cells during the cell line selection studies. The variant was initially observed as an atypical post-monomer fragment peak in size exclusion chromatography. Characterization of the fragment peak using intact and reduced liquid chromatography-mass spectrometry (LC-MS) analyses determined that the fragment consisted of a normal light chain disulfide-linked to an aberrant 26 kDa fragment that could not be assigned to any HC fragment even after considering common modifications. Further analysis using LC-MS/MS peptide mapping revealed that the aberrant fragment contained the expected HC amino acid sequence (1-232) followed by a 20-mer novel sequence corresponding to expression of heavy chain DNA sequence in the -1 reading frame. Examination of the DNA sequence around the frameshift initiation site revealed that a mononucleotide repeat GGGGGG located in the IgG1 HC constant region was most likely the structural root cause of the frameshift. Rapid identification of the frameshift allowed us to avoid use of a problematic cell line containing the frameshift as the production cell line. The frameshift reported here may be observed in other mAb products and the hypothesis-driven analytical approaches employed here may be valuable for rapid identification and characterization of frameshift variants in other recombinant proteins.     

Rapid comparison of a candidate biosimilar to an innovator monoclonal antibody with advanced liquid chromatography and mass spectrometry technologies

This study shows that state-of-the-art liquid chromatography (LC) and mass spectrometry (MS) can be used for rapid verification of identity and characterization of sequence variants and posttranslational modifications (PTMs) for antibody products. A candidate biosimilar IgG1 monoclonal antibody (mAb) was compared in detail to a commercially available innovator product. Intact protein mass, primary sequence, PTMs and the micro-differences between the two mAbs were identified and quantified simultaneously. Although very similar in terms of sequences and modifications, a mass difference observed by LC-MS intact mass measurements indicated that they were not identical. Peptide mapping, performed with data independent acquisition LC-MS using an alternating low and elevated collision energy scan mode (LC-MS^E), located the mass difference between the biosimilar and the innovator to a two amino acid residue variance in the heavy chain sequences. The peptide mapping technique was also used to comprehensively catalogue and compare the differences in PTMs of the biosimilar and innovator mAbs. Comprehensive glycosylation profiling confirmed that the proportion of individual glycans was different between the biosimilar and the innovator, although the number and identity of glycans were the same. These results demonstrate that the combination of accurate intact mass measurement, released glycan profiling and LC-MS^E peptide mapping provides a set of routine tools that can be used to comprehensively compare a candidate biosimilar and an innovator mAb.Key words: biosimilar mAb, innovator mAb, molecular similarity, sequence variants, posttranslational modifications, N-linked glycosylation, chemical degradations, micro-heterogeneities, characterization, intact protein mass measurement, peptide mapping, glycan profiling, LC-MS, LC-fluorescence, MALDI MS     

Cloning, high-level expression, single-step purification, and binding activity of His6-tagged recombinant type B botulinum neurotoxin heavy chain transmembrane and binding domain

Botulinum neurotoxins (BoNTs) are highly potent toxins that inhibit neurotransmitter release from peripheral cholinergic synapses and associate with infant botulism. BoNT is a approximately 150kDa protein, consisting of a binding/translocating heavy chain (HC; 100kDa) and a toxifying light chain (LC; 50kDa) linked through a disulfide bond. C-terminal half of the heavy chain is binding domain, and N-terminal half of the heavy chain is translocation domain that includes transmembrane domain. A functional botulinum neurotoxin type B heavy chain transmembrane and binding domain (Ile 624-Glu 1291) has been cloned into a bacterial expression vector pET 15b and produced as an N-terminally six-histidine-tagged fusion protein (BoNT/B HC TBD). (His(6))-BoNT/B HC TBD was highly expressed in Escherichia coli BL21-CodonPlus (DE3)-RIL and isolated from the E. coli inclusion bodies. After solubilizing the purified inclusion bodies with 6M guanidine-HCl in the presence of 10mM beta-mercaptoethanol, the protein was purified and refolded in a single step on Ni(2+) affinity column by removing beta-mercaptoethanol first, followed by the removal of urea. The purified protein was determined to be 98% pure as assessed by SDS-polyacrylamide gel. (His(6))-BoNT/B HC TBD retained binding to synaptotagmin II, the receptor of BoNT/B, which was confirmed by immunological dot blot assay, also to ganglioside, which was investigated using enzyme-linked immunosorbent assay.     

Rapid comparison of a candidate biosimilar to an innovator monoclonal antibody with advanced liquid chromatography and mass spectrometry technologies

This study shows that state-of-the-art liquid chromatography (LC) and mass spectrometry (MS) can be used for rapid verification of identity and characterization of sequence variants and posttranslational modifications (PTMs) for antibody products. A candidate biosimilar IgG1 monoclonal antibody (mAb) was compared in detail to a commercially available innovator product. Intact protein mass, primary sequence, PTMs, and the micro-differences between the two mAbs were identified and quantified simultaneously. Although very similar in terms of sequences and modifications, a mass difference observed by LC-MS intact mass measurements indicated that they were not identical. Peptide mapping, performed with data independent acquisition LC-MS using an alternating low and elevated collision energy scan mode (LC-MS^E), located the mass difference between the biosimilar and the innovator to a two amino acid residue variance in the heavy chain sequences. The peptide mapping technique was also used to comprehensively catalogue and compare the differences in PTMs of the biosimilar and innovator mAbs. Comprehensive glycosylation profiling confirmed that the proportion of individual glycans was different between the biosimilar and the innovator, although the number and identity of glycans were the same. These results demonstrate that the combination of accurate intact mass measurement, released glycan profiling, and LC-MS^E peptide mapping provides a set of routine tools that can be used to comprehensively compare a candidate biosimilar and an innovator mAb.     

Cloning, expression, and purification of C-terminal quarter of the heavy chain of botulinum neurotoxin type A

Botulinum neurotoxins (BoNTs) are highly potent toxins that inhibit neurotransmitter release from peripheral cholinergic synapses. BoNTs consist of a toxifying light chain (LC; 50 kDa) and a binding-translocating heavy chain (HC; 100 kDa) linked through a disulfide bond. The complete sequence of BoNT/A consists of 1296 amino acid residues. The beta-trefoil domain for BoNT/A to which gangliosides bind starts at Ser 1092 and this fragment represents the C-half of the C-terminus of the heavy chain (C-quarter HC or HCQ). The recombinant HCQ DNA was successfully cloned into an expression vector (pET15b), which was used to transform Escherichia coli strain BL21-Star (DE3) for expression. Expression of HCQ was obtained by an extended post-induction time of 15 h at 30 degrees C. The recombinant histidine tagged HCQ protein was isolated and purified by nickel affinity gel column chromatography and its molecular weight was verified by gel electrophoresis. The HCQ was positively identified by antibodies raised against BoNT/A employing immunological dot-blot and Western blot assays. HCQ was shown to bind with synaptotagmin (a known BoNT/A receptor) and gangliosides, indicating that the expressed and purified HCQ protein retains a functionally active conformation.     

Analysis of human antibody IgG2 domains by reversed-phase liquid chromatography and mass spectrometry

It has been well documented that papain cleaves an IgG1 molecule to release Fab and Fc domains; however, papain was found unable to release such domains from an IgG2. Here we present a new combinatory strategy to analyze the heterogeneity of the light chain (LC), single chain Fc (sFc), and Fab portion of the heavy chain (Fd) of an IgG2 molecule released by papain cleavage under mild reducing conditions. These domains were well separated on reversed-phase high performance liquid chromatography (RP-HPLC) and analyzed by in-line liquid chromatography time-of-flight mass spectrometry (LC–TOF/MS). In addition, some modifications of these domains were revealed by in-line mass spectrometry, and confirmed by the peptide mapping on LC–MS/MS analysis. This same strategy was proven suitable for IgG1 molecules as well. This procedure provides a simplified approach for the characterization of antibody biomolecules by facilitating the detection of low-level modifications in a domain. In addition, the technique offers a new strategy as an identification assay to distinguish IgG2 molecules on RP-HPLC, by which highly conserved Fc domains remain at a constant retention time (RT) unique to its subisotype, while varying RTs of the light chain and the Fd distinguish the monoclonal antibody from other molecules of the same isotype based on the underlying characteristics of each antibody.     

High Yield Preparation of Functionally Active Catalytic-Translocation Domain Module of Botulinum Neurotoxin Type A That Exhibits Uniquely Different Enzyme Kinetics

Botulinum neurotoxins (BoNTs) are the most toxic proteins known to cause flaccid muscle paralysis as a result of inhibition of neurotransmitter release from peripheral cholinergic synapses. BoNT type A (BoNT/A) is a 150 kDa protein consisting of two major subunits: light chain (LC) and heavy chain (HC). The LC is required for the catalytic activity of neurotoxin, whereas the C and N terminal domains of the HC are required for cell binding, and translocation of LC across the endosome membranes, respectively. To better understand the structural and functional aspects of BoNT/A intoxication we report here the development of high yield Escherichia coli expression system (2–20-fold higher yield than the value reported in the literature) for the production of recombinant light chain-translocation domain (rLC-TD/A) module of BoNT/A which is catalytically active and translocation competent. The open reading frame of rLC-TD/A was PCR amplified from deactivated recombinant BoNT/A gene (a non-select agent reagent), and was cloned using pET45b (+) vector to express in E. coli cells. The purification procedure included a sequential order of affinity chromatography, trypsinization, and anion exchange column chromatography. We were able to purify?>?95% pure, catalytically active and structurally well-folded protein. Comparison of enzyme kinetics of purified LC-TD/A to full-length toxin and recombinant light chain A suggest that the affinity for the substrate is in between endopeptidase domain and botulinum toxin. The potential application of the purified protein has been discussed in toxicity and translocation assays.     

A plasmid system for optimization of Fab' production in Escherichia coli: importance of balance of heavy chain and light chain synthesis

We demonstrate the importance of optimizing the balance of light chain (LC) and heavy chain (HC) expression to achieve high level production of Fab' fragments in the Escherichia coli periplasm. The LC:HC balance has been controlled by varying the codon usage of the signal peptide (SP) and 5' mature domain coding regions. Different SP coding regions have been identified from a codon wobble-based library using alkaline phosphatase (AP) as a reporter gene. A plasmid system that enables random combination of these variant SP coding regions is used to construct optimized Fab' expression plasmids. These small plasmid libraries facilitated selection of optimal Fab' expression plasmids and resulted in increases of periplasmic yield, up to 580 mgL(-1) from E. coli fermentations and will enable rapid variable region subcloning and selection of future Fab(') expression plasmids.     

Genetic and biochemical analysis of an endonuclease encoded by the IncN plasmid pKM101.

The IncN plasmid pKM101 nuc gene encodes a periplasmically localized endonuclease. DNA sequence analysis indicates that this gene encodes a hydrophilic protein of about 19.5 kDa containing a hydrophobic signal sequence. nuc is homologous to a partially sequenced open reading frame adjacent to the sog gene of the plasmid CollB-P9, a plasmid known to encode an endonuclease similar to that of pKM101. A partially sequenced tra gene directly upstream of nuc is homologous to the virB11 gene of Agrobacterium tumefaciens. We have partially purified the pKM101 nuclease by osmotic shock and cation exchange chromatography, and used this enzyme preparation to sequence the protein's amino terminus. The first 13 amino acids of the mature protein match amino acids 23 to 35 of the predicted sequence, indicating that the protein is proteolytically processed to a molecular mass of approximately 17 kDa, probably during export to the periplasmic space. The enzyme was able to attack many sites along an end labelled duplex DNA substrate, but showed clearly preferred cleavage sites, and may cleave preferentially at purine-rich regions.     

Identification of Bilirubin Binding Site in Type I Collagen

The interaction of bilirubin with collagen in the significance of jaundice incidence have been previously reported and investigated. The novel peptide sequences containing bilirubin binding domain was identified and located to develop a basis for further studies investigating the interactions of collagen with bilirubin in the present study. In this study an intricate interaction between bilirubin and collagen was characterized and their binding domain has been established using in-gel digestion and LC–MS/MS analysis based on the collagen sequencing and peptide mass fingerprinting. The biotinylated bilirubin derivatives bind to α₁(I) chain but not to α₂(I) chains which clearly designates that bilirubin shows greater affinity to α₁ chains of collagen. The intact proteins collected after analyzing the resulting complex mixture of peptides was used for peptide mapping. Using the electrospray method, among the other peptide sequence information obtained, the molecular weight of collagen alpha-2(I) chain was obtained by locating a 130 kDa weight peptide sequences with greater pi value (9.14) with 1,364 amino acid residues and collagen alpha-1(I) chain with 1,463 amino acid residues with 138.9 kDa molecular weight. This information leads to locate the exact sequence of these helices focussing on the domain identification. The total charge of the peptide domain sequences infers that the bilirubin participates in the electrostatic mode of interaction with collagen peptide. Moreover, other modes of interactions such as hydrogen bonding, covalent interactions and hydrophobic interactions are possible.     

重组人胰高血糖素样肽－1的表达及生物学活性

将人工合成的人胰高血糖素样肽-1（human glucagon like peptide-1, hGLP-1）基因插入质粒载体pET-32a(+)中,构建成rhGLP-1与硫氧还蛋白（thioredox）及六聚组氨酸（hexahistidine）的融合表达载体pET32-GLP-1,转化大肠杆菌BL21（DE3）获得表达菌株,经IPTG诱导发酵的菌体超声破碎后,裂解液用Ni离子亲和层析纯化得到融合蛋白,经肠激酶裂解,再次Ni离子亲和层析,得到rhGLP-1样品。经SDS PAGE 和等电聚焦检测,样品纯度大于90％, 等电点介于pH5.2～pH5.85之间。质谱测定rhGLP-1分子量为3 355.0kDa,肽图分析得到2 097.7kDa和1 005.5kDa两个胰蛋白酶酶解片断,均与理论分析结果一致。动物实验表明重组蛋白具有明显的降血糖活性和促胰岛素分泌作用。     

Mass spectrometric characterization of the human androgen receptor ligand-binding domain expressed in Escherichia coli

The ligand-binding domain (LBD) of the human androgen receptor (hAR LBD), encompassing amino acids (AAs) 647-919, was expressed in Escherichia coli with an N-terminal polyhistidine tag (His(10)-hAR LBD) from a pET-16b vector. The overexpressed protein was initially insoluble in inclusion bodies, and was subsequently solubilized in 8 M guanidine hydrochloride (GdnHCl). The solubilized His(10)-hAR LBD was purified to apparent homogeneity by metal ion affinity chromatography in the presence of 6 M GdnHCl. The isolated protein migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular mass of 33-34 kDa, as expected from the plasmid construct. Immunoblot analysis with C-terminal antibodies raised against a peptide corresponding to the last 19 AAs (AAs 901-919) of hAR revealed that the purified protein contained an immunoreactive epitope present within the AR and was of the appropriate size. Further characterization, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS), showed a single protein species of average mass 34 580 Da, confirming the size and purity of the purified His(10)-hAR LBD. Detailed tryptic peptide mapping analysis, using MALDI/TOF-MS, identified a total of eight peptides with a 30% coverage of the LBD, including the last tryptic peptide in the hAR sequence. These data confirm that the purified protein was the intact hAR LBD. AA sequencing of these tryptic peptides, using an HPLC-coupled electrospray ionization ion trap mass spectrometer (LC/ESI-ITMS and MS/MS), unambiguously confirmed that the peptides were from the hAR LBD. The purified His(10)-hAR LBD in 6 M GdnHCl could be renatured as determined by ligand-binding activity, with a similar equilibrium dissociation constant (K(d)) for [(3)H]-mibolerone and a similar steroid specificity to the AR isolated from rat ventral prostate.     

Mass spectrometric analysis of innovator,counterfeit, and follow‐on recombinant human growth hormone

We have performed a detailed characterization of recombinant human growth hormone that included the identification of the entire sequence with disulfide linkages as well as subtle modifications by a sensitive liquid chromatography coupled online with tandem mass spectrometry (LC‐MS) approach using the accurate peptide mass (FTICR MS) and sequence assignment (MS/MS measurement). The extent of oxidation, deamidation, and chain cleavages were measured by the ratio of peak areas of the nonmodified peptide vs. the sum of peak area of the nonmodified and modified peptides in the same LC‐MS analysis. The subtle but distinct differences were found in the recombinant human growth from the three manufacturers (the follow‐on, counterfeit, and the original innovator products). In relative comparison, the follow‐on product had the highest degree of oxidation at methionine residues, followed by the counterfeit product, and the original innovator product had the least amount of oxidation at all three sites with the similar oxidation order. In cases, the oxidation order was Met14 > Met125 > Met170. In contrast, the follow‐on had the least amount of deamidation at aspargine (Asn149), and the counterfeit had the highest degree of deamidation at this site. For the chain cleavage, the follow‐on product had the highest cleavage occurring at T 10 peptide (between Asn99 and Ser100), the counterfeit had the highest cleavage on T4 peptide, (between Glu30 and Phe31), and the original innovator product with the least amount of cleavages on both sites. These subtle but distinct differences are likely because of nonidentical manufacturing, formulation procedures, and storage conditions. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Isolation and characterization of two molecular variants of myosin heavy chain from rabbit ventricle. Change in their content during normal growth and after treatment with thyroid hormone

We have prepared monoclonal antibodies specific for cardiac myosin heavy chain. These antibodies were used for the separation and characterization of the molecular variants of myosin heavy chain present in the rabbit heart. Two molecular forms of myosin heavy chain, HC alpha and HC beta, were isolated from the euthyroid rabbit heart by affinity chromatography. Their reactivity with our antibodies indicated that the primary structures of HC alpha and HC beta differ in at least four and share at least two antigenic determinants. Differences in the primary structure of HC alpha and HC beta were confirmed by analysis of the peptides produced by limited chymotryptic digestion of the two heavy chains. Thirteen peptide differences were consistently found. The HC alpha and HC beta variants are shown by immunologic analysis and in chymotryptic peptide profiles to be identical with the predominant forms of myosin heavy chain synthesized in the hearts of hyperthyroid and adult euthyroid rabbits, respectively. During development and maturation of the euthyroid rabbit heart, HC alpha comprises approximately 50% of the ventricular myosin between birth and 4 weeks of age; it diminishes to 20-30% by 8 weeks and to 10-20% by 12 weeks of age. Cardiac myosin from a 1-year-old rabbit is composed almost entirely of HC beta. Cardiac myosin from embryonic animals at 20 days gestation contained 20% HC alpha. These results show that HC alpha occurs normally in the euthyroid rabbit heart and that the relative proportions of HC alpha and HC beta depend on both the developmental stage and the thyroid state of the animal.     

Light chain 1 from the Chlamydomonas outer dynein arm is a leucine-rich repeat protein associated with the motor domain of the gamma heavy chain.

The LC1 light chain from Chlamydomonas outer arm dynein is tightly bound to the gamma heavy chain. Molecular cloning revealed that LC1 is a member of the SDS22+ subclass of the leucine-rich repeat protein family and as such is likely involved in mediating interactions between dynein and the components of a signal transduction pathway. Through the combination of covalent cross-linking and vanadate-mediated photolysis, LC1 was found to associate with that portion of the gamma HC that is C-terminal to the P1 loop. This region comprises most of the globular head domain of the heavy chain and includes the stalk-like structure that is involved in microtubule binding. Attachment of LC1 to this region represents the only known example of an accessory polypeptide directly associated with a dynein motor domain. Additional cross-linking experiments revealed that LC1 also interacts directly in situ with an approximately 45 kDa axonemal component; this interaction is disrupted by the standard high salt treatment used to remove the outer arm from the axoneme. These data suggest that LC1 acts to mediate the association between this 45 kDa axonemal polypeptide and the motor unit of the gamma HC.     

Light Chain Separated from the Rest of the Type A Botulinum Neurotoxin Molecule Is the Most Catalytically Active Form

Botulinum neurotoxins (BoNT) are the most potent of all toxins. The 50 kDa N-terminal endopeptidase catalytic light chain (LC) of BoNT is located next to its central, putative translocation domain. After binding to the peripheral neurons, the central domain of BoNT helps the LC translocate into cytosol where its proteolytic action on SNARE (soluble NSF attachment protein receptor) proteins blocks exocytosis of acetyl choline leading to muscle paralysis and eventual death. The translocation domain also contains 105 Å -long stretch of ∼100 residues, known as “belt,” that crosses over and wraps around the LC to shield the active site from solvent. It is not known if the LC gets dissociated from the rest of the molecule in the cytosol before catalysis. To investigate the structural identity of the protease, we prepared four variants of type A BoNT (BoNT/A) LC, and compared their catalytic parameters with those of BoNT/A whole toxin. The four variants were LC + translocation domain, a trypsin-nicked LC + translocation domain, LC + belt, and a free LC. Our results showed that K_m for a 17-residue SNAP-25 (synaptosomal associated protein of 25 kDa) peptide for these constructs was not very different, but the turnover number (k _cat) for the free LC was 6-100-fold higher than those of its four variants. Moreover, none of the four variants of the LC was prone to autocatalysis. Our results clearly demonstrated that in vitro, the LC minus the rest of the molecule is the most catalytically active form. The results may have implication as to the identity of the active, toxic moiety of BoNT/A in vivo.     

The C-terminus of botulinum neurotoxin type A light chain contributes to solubility, catalysis, and stability

Botulinum neurotoxin type A (BoNT/A) is the etiological agent responsible for botulism, a disease characterized by peripheral neuromuscular blockade. BoNT/A is produced by Clostridium botulinum as a single chain protein that is activated by proteolytic cleavage to form a 50 kDa light chain (LC, 448 amino acids) and a disulfide bond-linked 100 kDa heavy chain (HC, 847 amino acids). Whilst HC comprises the receptor binding and translocation domains, LC is a Zn2+-endopeptidase that cleaves at a single glutaminyl-arginine bond corresponding to residues 197 and 198 at the C-terminus of SNAP25. Cleavage of SNAP25 uncouples the neural exocytosis docking/fusion machinery. LC/A (LC 1-448) and several C-terminal deletion proteins of LC/A were engineered and expressed as His-tagged fusion proteins in Escherichia coli. LC 1-448 was purified, but precipitated upon storage. Approximately 40% of LC 1-448 was a covalent dimer due to the formation of inter-chain disulfide bond formation at Cys430. Conversion of Cys430 to Ser abolished dimer formation of LC 1-448, but did not improve solubility. Three C-terminal deletion peptides were engineered; LC 1-425 and LC 1-418 were expressed and could be purified as soluble and stable proteins, whilst LC 1-398 was soluble, but not stable to storage. Kinetic studies showed that LC 1-448 and LC 1-425 efficiently cleaved GST-SNAP25 and the fluorescent substrate SNAPtide, while LC 1-418 catalyzed the cleavage of both the SNAP25 and the fluorescent substrate SNAPtide with a similar Km, but at a 10-fold slower kcat. Thus, regions within the C-terminus of LC/A contribute to solubility, stability, and catalysis.     

Expression, purification, and characterization of the recombinant kringle 1 domain from tissue-type plasminogen activator.

A novel fusion protein expression plasmid that allows ready purification and subsequent facile release of the target molecule has been constructed and employed to express in Escherichia coli and purify the tissue plasminogen activator kringle 1 domain ([K1tPA] residues C92-C173). The resulting plasmid encodes the tight lysine-binding kringle (K)1 domain of human plasminogen ([K1HPg]) followed by a peptide (PfXa) containing a factor Xa-sensitive bond, downstream of which [K1tPA] was inserted. The recombinant (r) [K1HPg]PfXa[K1tPA] fusion polypeptide was purified from various cell fractions in one step by Sepharose-lysine affinity chromatography. After cleavage with fXa, the mixture was repassaged over Sepharose-lysine, whereupon the r-[K1tPA]-containing polypeptide passed unretarded through the column. A homogeneous preparation of this material was then obtained after a simple step employing fast protein liquid chromatography. The purified r-[K1tPA], which contained the amino acid sequence SNAS[K1tPA]S, provided an amino-terminal amino acid sequence, through at least 20 amino acid residues, that was identical to that predicted from the cDNA sequence. The molecular mass of r-SNAS[K1tPA]S, determined by electrospray mass spectrometry, was 9621.9 +/- 4.0 (expected molecular mass, 9623.65). 1H-NMR spectroscopy and thermal stability studies of r-SNAS[K1tPA]S revealed that the purified material was properly folded and similar to other isolated kringle domains. Additionally, employment of this methodology revealed that only a very weak interaction between epsilon-aminocaproic acid and the isolated r-[K1tPA] domain occurred.