Sulakshana Jain

Immobilized metal affinity chromatography without chelating ligands: purification of soybean trypsin inhibitor on zinc alginate beads.

Immobilized metal affinity chromatography (IMAC) is a widely used technique for bioseparation of proteins in general and recombinant proteins with polyhistidine fusion tags in particular. An expensive and critical step in this process is coupling of a chelating ligand to the chromatographic matrix. This chelating ligand coordinates metal ions such as Cu2+, Zn2+, and Ni2+, which in turn bind proteins. The toxicity of chemicals required for coupling and their slow release during the separation process are of considerable concern. This is an important issue in the context of purification of proteins/enzymes which are used in food processing or pharmaceutical purposes. In this work, a simpler IMAC design is described which should lead to a paradigm shift in the application of IMAC in separation. It is shown that zinc alginate beads (formed by chelating alginate with Zn2+ directly) can be used for IMAC. As “proof of concept”, soybean trypsin inhibitor was purified 18‐fold from its crude extract with 90% recovery of biological activity. The dynamic binding capacity of the packed bed was 3919 U mL‐1, as determined by frontal analysis. The media could be regenerated with 8 M urea and reused five times without any appreciable loss in its binding capacity.

Polyelectrolyte Coated Multilayered Liposomes (Nanocapsules) for the Treatment of Helicobacter pylori Infection

Helicobacter pylori infection is one of the major causes of gastric cancers. A number of systems have already been reported, but 100% eradication has never been achieved. The present invention designs a gastro-retentive drug delivery system incorporated with amoxicillin and metronidazole, specifically suited for the eradication of Helicobacter pylori infections due to its mucoadhesiveness in the presence of polyelectrolyte polymers. The system possesses the advantages of both vesicular and particulate carriers, and it was prepared by alternative coating of polyanion (poly(acrylic acid), PAA) and polycation (poly(allylamine hydrochloride), PAH) using liposomes as the core. Compared with the conventional liposomes, the polyelectrolyte based multilayered system (nanocapsules) gave prolonged drug release in simulated gastric fluid, which is well suited for drug delivery against H. pylori infection in the stomach. In vitro growth inhibition study, agglutination assay, and in situ adherence assay in cultured H. pylori suggested the successful in vitro activity and binding propensity of the system. In vivo bacterial clearance study carried out in a H. pylori infected mouse model finally confirmed the success of the developed novel nanocapsule system. Thus, the newly developed composite nanocapsules along with the use of combination therapy proved to have commendable potential in Helicobacter pylori eradication as compared to already existing conventional and novel drug delivery systems.

Purification of goat immunoglobulin G by immobilized metal‐ion affinity using cross‐linked alginate beads

The alginate beads obtained by cross‐linking of the polymer by epichlorohydrin were charged with Cu(II). The copper‐charged beads could be directly used as a immobilized‐metal‐affinity‐chromatographic medium for purification of goat IgG. The best results in the packed‐bed mode were obtained by using beads charged with 95.4 μmol/ml Cu(II). We found that we could recover 97.4% IgG with an 8‐fold purification.

Evaluation of microbeads of calcium alginate as a fluidized bed medium for affinity chromatography of Aspergillus niger Pectinase.

Calcium alginate microbeads (212–425 μm) were prepared by spraying 2% (w/v) alginate solution into 1 M CaCl2 solution. The fluidization behavior of these beads was studied, and the bed expansion index and terminal velocity were found to be 4.3 and 1808 cm h−1, respectively. Residence time distribution curves showed that the dispersion of the protein was much less with these microbeads than with conventionally prepared calcium alginate macrobeads when both kinds of beads were used for chromatography in a fluidized bed format. The fluidized bed of these beads was used for the purification of pectinase from a commercial preparation. The media performed well even with diluted feedstock; 90% activity recovery with 211‐fold purification was observed.

A microassay for protein determination using microwaves

Recent developments in biochemistry demand faster screening methods which can work with small volumes. This is equally true for estimation of proteins as well. Akins and Tuan [1] have suggested the use of microwaves for enhancing the sensitivity of protein estimation by Lowry s method [2]. Young et al. [3] have described an automated colorimetric microassay system for protein estimation using an ELISA plate reader. In the present work, we describe a method which is based upon synergy of the above two approaches. The result is a fast and sensitive protocol which can work with small volumes.

Emerging options in protein bioseparation

Publisher Summary The chapter describes the way downstream processing scientists are meeting protein separation challenges. There is a distinct trend towards simultaneous purification and immobilization strategies. Leads from affinity precipitation have led to smart biocatalyst design which has inbuilt purification step. High performance biocatalyst designs such as cross-linked enzyme aggregate (CLEA) also do not require highly purified enzymes as starting material. Therefore, first one has had integration of upstream and downstream processes. Now, one has integration of purification and immobilization. Thus, there are enough reasons to believe that need (for efficient separation strategies) would continue to breed novel and innovative technologies.

Applications of alginate in bioseparation of proteins.

Alginate is a polysaccharide that is a block polymer consisting of block units of guluronic acid and mannuronic acid. It shows inherent biological affinity for a variety of enzymes such as pectinase, lipase, phospholipase D, α and β amylases and glucoamylase. Taking advantage of its precipitation with Ca2+ and the above-mentioned property, alginate has been used for purification of these enzymes by affinity precipitation, aqueous two phase separation, macroaffinity ligand facilitated three phase partitioning, immobilized metal affinity chromatography and expanded bed affinity chromatography. Thus, this versatile marine resource has tremendous potential in bioseparation of proteins.

An integrated process for separation of major and minor proteins from goat serum.

Analysis of minor proteins in animal sera is of considerable clinical significance. To be able to detect these proteins, depletion of major proteins (albumin and immunoglobulin G [IgG]) is necessary. Many of these proteins are also required in pure form for a variety of biochemical applications. The present work uses goat serum as the system and describes the separation and purification of both major and several minor proteins. This was carried out by judicious adaptation and combination of separation technologies such as immobilized metal ion affinity chromatography (on a somewhat novel matrix), dye affinity chromatography, and lectin affinity chromatography. Albumin, IgG, α2-macroglobulin, α1-proteinase inhibitor, and transferrin were obtained from the serum. The purified preparations were found to be homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis.

A Smart Bioconjugate of Trypsin with Alginate

Alginate is a polymer of guluronic acid and mannuronic acid residues and is an inexpensive, nontoxic polysaccharide of marine origin. Trypsin was immobilized noncovalently on alginate with 100% retention of activity. The enzyme did not leach off the polymer even in the presence of 0.01 M HCl and Triton X-100 (0.2% vv−1). The Vmax/Km values did not change significantly on immobilization. There was 22% loss of activity in first cycle of pH change and after that the conjugate could be reused upto 4 precipitation cycles without any further loss of activity. This smart bioconjugate was also found to have better operational stability in the presence of casein than free enzyme. Fluorescence studies were carried out to probe structural changes upon immobilization.