Erik Holmgren

An evaluation of different enzymatic cleavage methods for recombinant fusion proteins, applied on des(1-3)insulin-like growth factor I.

Different enzymatic methods for cleavage of recombinant fusion proteins were compared. To find an efficient cleavage method, five different fusion proteins were produced. The fusion proteins differed only in the linker region between the fusion partner and the desired product, human des(1–3)insulin-like growth factor I. A cleavage study was performed with enterokinase, plasmin, thrombin, urokinase, and recombinant H64A subtilisin. Significant cleavage was obtained using thrombin, H64A subtilisin, and enterokinase. Thrombin cleavage was studied on a larger scale and des(1–3)IGF-I was recovered at a final yield of 3 mg/L growth medium. Thrombin and enterokinase were also studied as immobilized proteases and they cleaved the fusion proteins with retained activity. To further improve thrombin cleavage, a continuous reactor was constructed, consisting of a closed system with a thrombin column and an ion exchange column in series. Here, the fusion protein circulated while free des(1–3)IGF-I was bound to the ion exchange column after release from the fusion protein. In the reactor, thrombin was as efficient as the free enzyme but gave a diminished rate of product degradation.

Anoctamin 2 identified as an autoimmune target in multiple sclerosis

Significance Despite the growing evidence that autoantibodies are team players in the pathogenesis of multiple sclerosis (MS), the target autoantigens are yet to be identified. In this work, we mined the autoantibody repertoire within MS by screening more than 2,000 plasma samples from patients with MS and controls and identified increased autoantibody reactivity against an ion-channel protein called “anoctamin 2” (ANO2). This finding points toward an ANO2 autoimmune sub-phenotype in MS and might contribute to the development of clinical algorithms to characterize a subgroup of MS patients. Multiple sclerosis (MS) is the most common chronic inflammatory disease of the central nervous system and also is regarded as an autoimmune condition. However, the antigenic targets of the autoimmune response in MS have not yet been deciphered. In an effort to mine the autoantibody repertoire within MS, we profiled 2,169 plasma samples from MS cases and population-based controls using bead arrays built with 384 human protein fragments selected from an initial screening with 11,520 antigens. Our data revealed prominently increased autoantibody reactivity against the chloride-channel protein anoctamin 2 (ANO2) in MS cases compared with controls. This finding was corroborated in independent assays with alternative protein constructs and by epitope mapping with peptides covering the identified region of ANO2. Additionally, we found a strong interaction between the presence of ANO2 autoantibodies and the HLA complex MS-associated DRB1*15 allele, reinforcing a potential role for ANO2 autoreactivity in MS etiopathogenesis. Furthermore, immunofluorescence analysis in human MS brain tissue showed ANO2 expression as small cellular aggregates near and inside MS lesions. Thus this study represents one of the largest efforts to characterize the autoantibody repertoire within MS. The findings presented here demonstrate that an ANO2 autoimmune subphenotype may exist in MS and lay the groundwork for further studies focusing on the pathogenic role of ANO2 autoantibodies in MS.

Differential stability of recombinant human insulin-like growth factor II in Escherichia coli and Staphylococcus aureus.

Recombinant human insulin-like growth factor II (IGF-II), produced as a soluble extracellular fusion protein, was shown to be proteolytically degraded in Escherichia coli. In contrast, the fusion protein secreted from Staphylococcus aureus was stable and the full length product could be recovered by affinity chromatography. After site specific cleavage of the fusion protein, soluble IGF-II with biological activity was obtained without refolding procedures. These results demonstrate that a eukaryotic protein unstable in E. coli can be stabilized by expression in a Gram positive host. The full-length fusion protein from S. aureus was used to characterize the protease responsible for the degradation in E. coli. Biochemical and genetic analysis suggests a specific degradation by the outer membrane protease (OmpT).

Designing a Multimer Allergen for Diagnosis and Immunotherapy of Dog Allergic Patients

Background Dog dander extract used for diagnosis and allergen-specific immunotherapy is often of variable and of poor quality. Objective To assemble four well-established dog allergen components into one recombinant folded protein for improved diagnosis and vaccination of allergy to dog. Methods A linked molecule, comprising the four dog lipocalin allergens Can f 1, Can f 2, Can f 4 and Can f 6 was constructed. The tetrameric protein was structurally characterized by small angle X-ray scattering, and compared with each single recombinant lipocalin allergen or an equimolar mix of the four allergens by analytical size exclusion chromatography, circular dichroism, allergen-specific IgE in serum by ELISA and allergen-dependent capacity to activate basophils. The immunogenicity of the fusion protein was evaluated in immunized mice by assessing splenocyte proliferation and antibody production. Results The linked tetrameric construct was produced as a soluble fusion protein, with the specific folds of the four individual allergens conserved. This multi-allergen molecule was significantly more efficient (p<0.001) than each single recombinant allergen in binding to dog-specific IgE, and the epitope spectrum was unaffected compared to an equimolar mix of the four allergens. Basophil degranulation revealed that the biologic activity of the linked molecule was retained. Immunization of mice with the linked construct induced comparable allergen-specific IgG responses with blocking capacity towards all included allergens and generated comparably low T-cell responses. Conclusion We provide the first evidence for a linked recombinant molecule covering the major dog allergens for potential use in diagnostics and allergy vaccination of dog allergic patients.

Synthesis and secretion of a fibrinolytically active tissue-type plasminogen activator variant in Escherichia coli

A gene encoding a variant (lacking amino acids 6-173) of human tissue-type plasminogen activator (t-PA), consisting only of the second kringle domain (K2) and the serine protease domain (P), was fused to a DNA segment coding for the signal peptide of staphylococcal protein A and a synthetic gene coding for a protein with ability to bind immunoglobulin G (IgG). The fusion protein which was synthesized in Escherichia coli and secreted into the growth medium, was found to be fibrinolytically active. Purification of the fusion protein was performed in a single step by affinity chromatography with immobilized IgG. Enzymatically active K2P was liberated from the fusion protein by cleavage at a unique Asn-Gly dipeptide sequence using hydroxylamine. These results demonstrate that a variant of human t-PA can be synthesized and secreted by E. coli as a fibrinolytically active fusion protein, which upon specific cleavage yields an active variant t-PA of the expected size.