Jan-Christer Janson

Agar derivatives for chromatography, electrophoresis and gel-bound enzymes: I. Desulphated and reduced cross-linked agar and agarose in spherical bead form

Abstract A method for producing alkali- and thermo-stable, insoluble spherical agar particles with very low adsorption capacity (tested with cytochrome C) is described. Sulphate ester groups present in the original agar are removed by alkaline hydrolysis of cross-linked agar, in bead form, at an elevated temperature. The reaction is performed in the presence of sodium borohydride to prevent simultaneous oxidation. Further reduction of the adsorption capacity may be accomplished by treatment of the gel with lithium aluminium hydride in dioxane. Desulphated and reduced agar and agarose gels can be packed in beds with excellent flow and molecular sieve properties.

Urea gradient size-exclusion chromatography enhanced the yield of lysozyme refolding.

Protein refolding is still a bottleneck for large-scale production of valuable proteins expressed as inclusion bodies in Escherichia coli. Usually biologically active proteins cannot be obtained with high yield at a high concentration after refolding. In order to meet the challenge of protein refolding a urea gradient gel filtration-refolding system was developed in this article. A Superdex 75 column was pre-equilibrated with a linear decreased urea gradient, the denatured protein experienced the gradual decrease in urea concentration as it went through the column. The refolding of denatured lysozyme showed this method could significantly increase the activity recovery of denatured lysozyme at high protein concentration. The activity recovery of 90% was obtained from the initial protein concentration up to 17 mg/ml within 40 min.

AGAR DERIVATIVES FOR CHROMATOGRAPHY, ELECTROPHORESIS AND GEL-BOUND ENZYMES III. RIGID AGAROSE GELS CROSS-LINKED WITH DIVINYL SULPHONE (DVS)

Agarose cross-linked with divinyl sulphone (DVS) is a new matrix for chromatography and immobilized enzymes that has distinct advantages over common agarose gels. It has outstanding mechanical stability as compared with these gels, and the rigid gel beads form beds permitting very high flow-rates. In addition, DVS-agarose is superior to agarose gels with respect to chemical stability in acid and neutral media. In alkaline solutions above pH 8, there is a slow elimination of the sulphone-containing bridges, but without noticeable concomitant dissolution of the gels below pH 12 for moderately or highly cross-linked gels. The DVS-agarose is sufficiently thermostable to be heated in an autoclave.

Studies on myrosinases: I. Purification and characterization of a myrosinase from white mustard seed (Sinapis alba, L.)

Myrosinases (EC 3.2.3.1) are glucosinolate (thioglucoside) hydrolases primarily occurring in plants of the Cruciferae family. 1. 1.|The separation of three myrosinase isoenzymes in Sinapis alba seed was achieved by DEAE-cellulose chromatography on Whatman DE-52. 2. 2.|The main myrosinase component was completely purified by DEAE-cellulose chromatography followed by gel chromatography on Sephadex G-200 and isoelectric focusing in an LKB-column. 3. 3.|The isolated myrosinase was found to be a glycoprotein with a molecular weight of 151 000, consisting of two identical polypeptide subunits with a molecular weight of 62 000 each and a carbohydrate part. The electrophoretic mobility was determined to 7.0·10−4 cm2/s per V and the isoelectric point was found to be pH 5.08.

endo-β-1,4-Mannanases from blue mussel, Mytilus edulis: purification, characterization, and mode of action

Two variants of an endo-β-1,4-mannanase from the digestive tract of blue mussel, Mytilus edulis, were purified by a combination of immobilized metal ion affinity chromatography, size exclusion chromatography in the absence and presence of guanidine hydrochloride and ion exchange chromatography. The purified enzymes were characterized with regard to enzymatic properties, molecular weight, isoelectric point, amino acid composition and N-terminal sequence. They are monomeric proteins with molecular masses of 39&#;216 and 39&#;265 Da, respectively, as measured by MALDI-TOF mass spectrometry. The isoelectric points of both enzymes were estimated to be around 7.8, however slightly different, by isoelectric focusing in polyacrylamide gel. The enzymes are stable from pH 4.0 to 9.0 and have their maximum activities at a pH about 5.2. The optimum temperature of both enzymes is around 50-55oC. Their stability decreases rapidly when going from 40 to 50oC. The N-terminal sequences (12 residues) were identical for the two variants. They can be completely renatured after denaturation in 6 M guanidine hydrochloride. The enzymes readily degrade the galactomannans from locust bean gum and ivory nut mannan but show no cross-specificity for xylan and carboxymethyl cellulose. There is no binding ability observed towards cellulose and mannan (Less)

Introduction to chromatography

A simple introduction to chromatography, considering thin-layer, column and gas-liquid methods, explaining the factors governing choice of one or the other. Basic references and suppliers of equipment are included.

Adsorption phenomena on sephacryl® S-200 superfine

Abstract The adsorption properties of Sephacryl S-200 are dependent upon the pH of the eluent buffer employed at the net surface charge of the solutes under investigation. At pH 3.5 the gel adsorbs acidic, neutral and basic proteins. By raising the pH of the eluent to 5.5 all adsorbed material can be desorbed from the gel. At pH 5.5 the gel showed no tendency to adsorb proteins of any surface charge. At this pH the gel acts as a passive molecular sieve. At pH 8 the gel begins to behave as a cation exchanger and strongly adsorbs any protein that is positively charged at this pH. Acidic or neutral proteins are eluted unretarded. These cation-exchange properties can be eliminated by including at least 0.2 M NaCl in the eluent buffer. At low ionic strength of the eluent buffer, DNA, tRNA and rRNA are adsorbed on to the gel at pH 3.5 or 5.5 but are completely desorbed by stepwise elution with 0.5 M NaCl in equilibrating buffer. The HETP values calculated for several solutes indicate that Sephacryl S-200 gives higher resolution and less zone-spreading than does Sephadex G-200.

Large-scale chromatography of proteins

Basic chromatography theory and its implication for scaling-up is thoroughly discussed as well as the implication of flow resistance in compressible gel materials. The advantages and disadvantages of different column designs are discussed, e.g. their effect on the non-chromatographic zone spreading. The properties of various column packing materials are compared and procedures for their maintenance are described. The engineering aspects of the construction of production scale columns are treated in detail as well as the operational behaviour of bed packings and the design of column end pieces. Aspects on the choice of pipe diameter, valve types and pumps for process chromatography are discussed and arrangements for filters, sampling devices, pressure switches etc. are suggested. Automation of chromatographic processes is briefly discussed. Examples of industrial applications of gel filtration, ion exchange chromatography and affinity chromatography are given, primarily within the pharmaceutical industry (plasma protein fractionation, insulin purification etc.).

On the history of the development of Sephadex

SummaryThe creative and stimulating atmosphere in the laboratories of The Svedberg and Arne Tiselius in the Departments of Physical Chemistry and Biochemistry of Uppsala University during the 1930s, 1940s and 1950s, was a nursery for a remarkable set of both academic and industrial advances. Their pupils were to become distinguished professors at Swedish Universities, as well as abroad, and they were directly involved in the development of two successful Swedish industries, LKB-produkter AB in Stockholm and Pharmacia AB in Uppsala. This review describes the preconditions and the events which led to the development of one of the first commercially available biochemical separation products, the gel filtration medium Sephadex which was introduced by Pharmacia AB in 1959. All the necessary components of a successful transfer of academic research to industrial product development were at hand: a scientific culture of common origin and a longstanding tradition in methodological research; mutual understanding and respect combined with informal links not only between the scientists involved but also between the president of the company and the university authorities. So far, 49 products (excluding those intended for use in health care and diagnostics) have been developed based on the epichlorohydrin cross-linked dextran gel Sephadex.

Delineation of a linear epitope by multiple peptide synthesis and phage display

Two different approaches, the phage display technique and the Spot peptide synthesis on cellulose membranes, were used to identify sequences recognized by Fab 57P, specific for tobacco mosaic virus protein (TMVP), and define the preferred chemical composition of a functional epitope. Kinetic measurements of the interaction between peptide variants and the antibody fragment were used to further refine the molecular basis of binding activity. Our results show that the functional epitope of Fab 57P requires precise physico-chemical properties at a limited number of positions, and that residues flanking these key residues can influence binding affinity. The phage display and Spot synthesis methods allowed the straightforward localization of the binding region and the identification of residues that are essential for recognition. However, these methods yielded slightly different views of accessory factors that are able to influence antibody binding. The influence on binding activity of these factors can only be assessed through quantitative affinity measurements.