Andrey N. Wulfson

Determination of indole alkaloids from R. serpentina and R. vomitoria by high-performance liquid chromatography and high-performance thin-layer chromatography

The separation of a model mixture of six indole alkaloids by high-performance liquid chromatography (HPLC) and high-performance thin-layer chromatography (HPTLC) on the normal- and reversed-phase Armsorb supports, respectively, was studied. A procedure for the analysis of extracts from plants cell callus culture of Rauwolfia serpentina and hairy roots of R. serpentina and R. vomitoria in the presence of these alkaloids and its derivatives with different chromatographic systems was elaborated. It was found that the alkaloid compositions of plant cell cultures and hairy roots of R. serpentina and R. vomitoria are identical. With HPLC analysis a higher precision of alkaloid determination in solution and measurements of concentration is achieved. HPTLC analysis gives a qualitative identification of the extracts. The combination of these high-performance methods provides quantitative and qualitative analyses of indole alkaloids such as ajmaline, ajmalicine, reserpine, raucaffricine, serpentine and yohimbine.

Recombinant human insulin: III. High-performance liquid chromatography and high-performance capillary electrophoresis control in the analysis of step-by-step production of recombinant human insulin☆

The production of recombinant human insulin consists of five main stages, accompanied by considerable transformation of molecules, concerning size, secondary structure and the presence of charged groups. The application of different methods, i.e., size-exclusion, ion-exchange and reversed-phase high-performance liquid chromatography (HPLC) and high-performance capillary electrophoresis (HPCE) (capillary zone electrophoresis and micellar electrokinetic capillary chromatography), to the analysis of insulin, insulin-related and non-insulin-related substances was studied. A combined HPLC-HPCE system for the step-by-step control of recombinant human insulin production technology is suggested. The advantages and shortcomings of these methods are discussed.

Bacterial production and refolding from inclusion bodies of a “Weak” toxin, a disulfide rich protein

The gene for the “weak” toxin of Naja kaouthia venom was expressed in Escherichia coli. “Weak” toxin is a specific inhibitor of nicotine acetylcholine receptor, but mechanisms of interaction of similar neurotoxins with receptors are still unknown. Systems previously elaborated for neurotoxin II from venom of the cobra Naja oxiana were tested for bacterial production of “weak” toxin from N. kaouthia venom. Constructs were designed for cytoplasmic production of N. kaouthia “weak” toxin in the form of a fused polypeptide chain with thioredoxin and for secretion with the leader peptide STII. However, it became possible to obtain “weak” toxin in milligram amounts only within cytoplasmic inclusion bodies. Different approaches for refolding of the toxin were tested, and conditions for optimization of the yield of the target protein during refolding were investigated. The resulting protein was characterized by mass spectrometry and CD and NMR spectroscopy. Experiments on competitive inhibition of 125I-labeled α-bungarotoxin binding to the Torpedo californica electric organ membranes containing the muscle-type nicotine acetylcholine receptor (α12β1γδ) showed the presence of biological activity of the recombinant “weak” toxin close to the activity of the natural toxin (IC50 = 4.3 ± 0.3 and 3.0 ± 0.5 µM, respectively). The interaction of the recombinant toxin with α7 type human neuronal acetylcholine receptor transfected in the GH4C1 cell line also showed the presence of activity close to that of the natural toxin (IC50 31 ± 5.0 and 14.8 ± 1.3 µM, respectively). The developed bacterial system for production of N. kaouthia venom “weak” toxin was used to obtain 15N-labeled analog of the neurotoxin.

A new hybrid protein for production of recombinant bacteriorhodopsin in Escherichia coli

Unique properties of bacteriorhodopsin, namely, photochromism and high thermal stability, make this protein an attractive target for physico-chemical studies, as well as for various biotechnological applications. Using Mistic as a suitable carrier for insertion of recombinant membrane proteins into cytoplasmic membrane of Escherichia coli, we developed a system for overexpression of bacteriorhodopsin and worked out an efficient procedure for its purification and renaturation with the final yield of 120 mg/l of refolded protein, which is the highest value reported to date for bacteriorhodopsin produced in E. coli. Functional activity of recombinant bacteriorhodopsin was confirmed by spectroscopic and electrochemical assays.

Recombinant human insulin IX. Investigation of factors, influencing the folding of fusion protein-S-sulfonates, biotechnological precursors of human insulin

The peculiarities of molecular structures and the influence of reaction conditions on the folding efficiency of fusion proteins-biotechnological precursors of human insulin, expressed in Escherichia coli as inclusion bodies have been investigated. The fusion proteins contained proinsulin sequence with various leader peptides connected by an Arg residue to the insulin B-chain. The kind and the size of leader peptide do not have essential influence on folding efficiency. However, the efficiency of protein folding depends on the location of the (His)6 site, which is used for metal-chelating affinity chromatography. In our study the protein folding depends on the reaction medium composition (including additives), the presence of accompanied cell components, pH, temperature, concentrations of protein, and redox agents. A negative influence of nucleic acid and heavy metal ions on folding has been found. S-sulfonated fusion protein has proinsulin-like secondary structure (by CD-spectroscopy data) that is the key point for 95% efficient folding proceeding. Folded fusion proteins are transformed into insulin by enzymatic cleavage.

Recombinant human insulin V Optimization of the reversed-phase high-performance liquid chromatographic separation

The applicability of reversed-phase high-performance liquid chromatography (HPLC) to the analysis of the products of recombinant insulin was studied. The influence of several mobile phases in reversed-phase and ion-pair HPLC on selectivity, resolution and sensitivity was investigated. Optimum conditions for the separation of insulin-related proteins on commercial and laboratory-made supports were established by means of three-dimensional optimizations of selectivity and resolution as a function of pH and ionic strength (mu). A mechanism for the separation of proteins with a mobile phase containing a high salt concentration and a pH near the isoelectric point of proteins is proposed. The questions of scaling up are considered. The proposed techniques allow the analysis of the main impurities and ensures a high quality of active insulin production.

A general approach to renaturation of recombinant proteins produced as inclusion bodies.

Renaturation is one of the most intensely studied problems in modern biochemistry. According to current views, the formation of the protein spatial structure (folding) in vivo proceeds in several stages via successive rendering to its conformation native. The first stage is a rapid accumulation of structured regions of the growing polypeptide chain during and after its biosynthesis. The second stage is formation of an intermediate known as the “molten globule” [1]. At the third (final) stage, the native protein structure determined by the amino acid sequence is completely formed and fixed. This stage is mediated by chaperonines and specific isomerases [2–4]. In the cell, the “molten globule” state is the start of interaction between the protein molecule and chaperonines, which provide the proper folding of the polypeptide chain. In the case of overexpression of an eukaryotic protein in a bacterial cell, its renaturation is often hampered, because chaperonines and auxiliary proteins of the prokaryotic cells cannot, or do not have enough time to, ensure the proper folding of the foreign protein. As a result, partially renatured protein with some elements of the secondary structure is accumulated in the cell [5] in the form of insoluble aggregates called inclusion bodies [6]. For this reason, the production of protein molecules with biologically active spatial structure requires in vitro renaturation, which is the key stage of protein production in biotechnology.

Receptor-binding domain of ephrin-A1: Production in bacterial expression system and activity

Eph receptor tyrosine kinases and their ligands, the ephrins, perform an important regulatory function in tissue organization, as well as participate in malignant transformation of cells. Ephrin-A1, a ligand of A class Eph receptors, is a modulator of tumor growth and progression, and the mechanism of its action needs detailed investigation. Here we report on the development of a system for bacterial expression of an ephrin-A1 receptor-binding domain (eA1), a procedure for its purification, and its renaturation with final yield of 50 mg/liter of culture. Functional activity of eA1 was confirmed by immunoblotting, laser scanning confocal microscopy, and flow cytometry. It is shown that monomeric non-glycosylated receptor-binding domain of ephrin-A1 is able to activate cellular EphA2 receptors, stimulating their phosphorylation. Ligand eA1 can be used to study the features of ephrin-A1 interactions with different A class Eph receptors. The created expression cassette is suitable for the development of ligands with increased activity and selectivity and experimental systems for the delivery of cytotoxins into tumor cells that overexpress EphA2 or other class A Eph receptors.

Recombinant human insulin: X simplification of folding of the biotechnological precursor of recombinant human insulin

The folding of biotechnological precursor of the human insulin precursor was carried out from solubilized inclusion bodies without a preliminary oxidizing or reducing its Cys residues. The inclusion bodies were dissolved in 8 M urea with the addition of 10 mM 2-mercaptoethanol. Hydrophobic cell components were removed from the solution by passing through a neutral weakly hydrophobic sorbent, the solution was five times diluted and refolded upon addition of 0.3 mM cystine for initiation of disulfide rearrangement. The presence of nucleic acids and cell protein impurities does not affect the folding efficiency. The resulting precursor of folded human insulin was purified by metal-chelate affinity chromatography and converted into insulin by two-stage enzymatic cleavage.

Recombinant human insulin VI. Determination of recombinant human proinsulin by capillary zone electrophoresis: optimization of efficiency and selectivity

The optimization of the separation of recombinant human insulin (rhI) and recombinant human proinsulin (rhP) by free-solution capillary zone electrophoresis in uncoated fused-silica capillaries with ionic and zwitterionic buffers was carried out. The relationship between the selectivity and pH of the buffer was established. The effects of pH and ionic strength of the buffer on protein adsorption on the capillary walls and Taylor diffusion was investigated. The separation of rhI and rhP with an efficiency of 200,000 theoretical plates was achieved using 20 mM Na2HPO4-NaOH buffer (pH 11.2). The proposed method allows the simultaneously determination of rhP and some rhI degradation products (which are also included in pharmacopoeias) in pharmacopoeially limited amounts in rhI.