Protein adsorption on histidyl-aminohexyl-Sepharose 4B: II. Application to the negative one-step affinity purification of human β2-microglobulin and Immunoglobulin G

The adsorption of two human proteins, β2-microglobulin and Immunoglobulin G, from uremic patient’s blood ultrafiltrate and plasma, respectively, was investigated on the histidyl-aminohexyl-Sepharose 4B adsorbent. Both target proteins could be adsorbed on the gel through a low affinity for immobilized histidine ligand. However, a fine adjustment of the operating conditions (ionic strength, buffer, pH) prevented their adsorption and thus allowed their “negative affinity” purification (purity estimated by silver nitrate SDS–PAGE) by the removal of the contaminating proteins. This simple and efficient method provides purification under gentle chromatographic conditions and a further characterization of both molecules.     

A novel core fractionation process of human plasma by expanded bed adsorption chromatography

Current plasma fractionation technology combines ethanol precipitation with packed bed chromatography. We have developed a novel core fractionation process comprising five expanded bed adsorption (EBA) chromatographic steps on high-density modified agarose/tungsten carbide beads. Plasma was first chromatographed on two diethyl amino-ethyl (DEAE)-tungsten carbide agarose adsorbents (respective mean particle diameters of d_v(0.5) = 190 and 37 μm) to isolate at 50 to 80% recovery a fraction containing 4 to 7 IU/ml factor II (FII), factor IX (FIX), and factor X (FX) (specific activity >1 IU/mg) and another enriched in FVIII and von Willebrand factor (vWF) (∼1 IU/ml and 0.6 IU/mg, respectively). The flow-through was adsorbed on 4% agarose-10% tungsten carbide beads coupled with an acidic mixed-mode ligand to isolate an 80% pure immunoglobulin G (IgG) at a 93% step recovery. A highly purified α1-antitrypsin was isolated at 95% step recovery by adsorbing the flow-through on 4% epoxy-crosslinked agarose-10% tungsten carbide adsorbent material coupled with a cationic ligand. Isolation of 98% pure albumin was achieved at a 99% step recovery by pH 4.5 adsorption of the flow-through on 6% agarose-10% tungsten carbide beads coupled with an acidic mixed-mode ligand. EBA may represent a feasible alternative core plasma fractionation tool.     

Phyletic relationships of protein structures based on spatial preference of residues

Summary A structure-based scoring matrix MDPRE was derived from amino acid spatial preferences in protein structures. Sequence alignment and evolutionary studies by using MDPRE matrix gave similar results as those from ordinary sequence and structure alignments. It is interesting that a matrix derived from structure data solely could give comparable alignment results, strongly indicating the intimate connection between protein sequences and structures. The branch order and length from this approach were close to those obtained by a structure comparison method. Thus, by applying this structure-based matrix, the trees obtained should reflect evolutionary characteristics of protein structure. This approach takes advantage over a direct structure comparison in that (1) only a sequence and MDPRE matrix are needed, making it simple and widely applicable (especially in the absence of 3-dimensional protein structure data); (2) an established algorithm for sequence alignment and tree building could be employed, providing opportunities for direct comparison between matrices from different methodologies. One of the most striking features of this method is its capability to detect protein structure homologies when the sequence identities are low. This was well reflected in the given examples of the alignment of dinucleotidebinding domains.     

Immobilized metal-ion affinity chromatography of human antibodies and their proteolytic fragments

Immobilized metal-ion affinity chromatography (IMAC) performed with four different transition metal ions: copper(II), nickel(II), zinc(II) and cobalt(II), was used to study the adsorption properties of human polyclonal gamma-globulines (IgG), Cohn II-III fractions, and their pepsin cleaved fragments: Fab'2 and F'c. In each case, digested products showed lower affinity for metal ions, as well by decreasing pH elution as by competition with imidazole. An explanation was proposed by the presence of a histidine (His) cluster in the F'c domain of IgGs, identified by computer calculation (accessible surface area (ASA) determination) as the more probable His 433-x-His 435 sequence presented in the CH3 domain of human IgG heavy chain. As shown by IMAC and electrophoresis, F'c and undigested IgG have higher affinity for transition metal ions than Fab'2 fragments and could be then separated in one step by IMAC. When chelated Zn(II) or Co(II) are used as ligands, the Fab'2 fragment could be easily recovered under mild conditions (pH 7) in the non-retained fraction. This approach could be used as a powerful alternative to conventional protein A/G methods for the commercial preparation of non immunogen active Fab'2 fragments.     

Purification of horse immunoglobulin isotypes based on differential elution properties of isotypes from protein A and protein G columns

Elution properties of horse immunoglobulin isotypes from protein A and protein G columns were examined. IgGa and IgGb isotypes were bound to protein A and protein G columns and were eluted by adjusting the pH of the elution buffer from 8.0 to 2.0. IgGc bound to protein G column but not to protein A column while IgG(T) bound to both columns. IgM and IgA apparently appeared not to bind to either column. New methods for purification of serum isotypes were developed using protein A and protein G columns as well as formerly established methods. Using these methods, it was possible to obtain purified isotypes for establishment of immunological assays for practical clinical use.     

Assessment of viral inactivation during pH 3.3 pepsin digestion and caprylic acid treatment of antivenoms

Antivenoms are manufactured by the fractionation of animal plasma which may possibly be contaminated by infectious agents pathogenic to humans. This study was carried out to determine whether pre-existing antivenom production steps, as carried out by EgyVac in Egypt, may reduce viral risks. Two typical manufacturing steps were studied by performing down-scaled viral inactivation experiments: (a) a pH 3.3 pepsin digestion of diluted plasma at 30 degrees C for 1h, and (b) a caprylic acid treatment of a purified F(ab')2 fragment fraction at 18 degrees C for 1h. Three lipid-enveloped (LE) viruses [bovine viral diarrhoea virus (BVDV), pseudorabies virus (PRV), and vesicular stomatitis virus (VSV)] and one non-lipid-enveloped (NLE) virus [encephalomyocarditis virus (EMC)] were used as models. Kinetics of inactivation was determined by taking samples at 3 time-points during the treatments. The pH 3.3 pepsin digestion resulted in complete clearance of PRV (>7.0 log(10)) and in almost complete reduction of VSV (>4.5 but < or =6.4 log(10)), and in a limited inactivation of BVDV (1.7 log(10)). EMC inactivation was > or =2.5 but < or =5.7 log(10). The caprylic acid treatment resulted in complete inactivation of the 3 LE viruses tested: BVDV (>6.6 log(10)), PRV (>6.6 log(10)), and VSV (>7.0 log(10)). For EMC no significant reduction was obtained (0.7 log(10)). Cumulative reduction was >13.6, >11.5, >8.3 and > or =2.5 for PRV, VSV, BVDV and EMC, respectively. Therefore the current manufacturing processes of at least some animal antisera already include production steps that can ensure robust viral inactivation of LE viruses and moderate inactivation of a NLE virus.     

Mannosylerythritol lipids: a review

Mannosylerythritol lipids (MELs) are surface active compounds that belong to the glycolipid class of biosurfactants (BSs). MELs are produced by Pseudozyma sp. as a major component while Ustilago sp. produces them as a minor component. Although MELs have been known for over five decades, they recently regained attention due to their environmental compatibility, mild production conditions, structural diversity, self-assembling properties and versatile biochemical functions. In this review, the MEL producing microorganisms, the production conditions, their applications, their diverse structures and self-assembling properties are discussed. The biosynthetic pathways and the regulatory mechanisms involved in the production of MEL are also explained here.     

FMMU白化豚鼠免疫学特性研究

目的　通过测定补体含量、免疫球蛋白含量、淋巴细胞的转化率及红细胞免疫功能 ,比较FMMU白化豚鼠和花色豚鼠的免疫学特性。方法　利用自动生化分析仪测定FMMU白化豚鼠和普通花色豚鼠的免疫球蛋白含量 (IgG、IgA、IgM)、C3、C4含量及总补体水平 ;利用淋巴细胞转化实验测定淋巴细胞的转化率 ;利用红细胞免疫复合物 (immunocomplex ,IC)花环形成试验测定两种豚鼠红细胞免疫粘附功能。结果　FMMU白化豚鼠血清中免疫球蛋白含量 (IgG、IgA、IgM)、C3、C4含量及补体总活性均显著低于花色豚鼠 ;FMMU白化豚鼠淋巴细胞转化率比花色豚鼠略低 ;FMMU白化豚鼠红细胞C3bR花环形成率与花色豚鼠差异无显著性 (P >0 0 5 )。而RBC IC花环率显著低于花色豚鼠 (P <0 0 5 )。结论　封闭群FMMU白化豚鼠具有独特的免疫学特性 ,总体免疫学功能低于花色豚鼠。