Dr. Vladimir F. Nesterenko, Institute of Biomedical Technologies, World Technology University, Moscow, Russia
Dr. Zhanna I. Andreeva, Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences
An instrument for quick choice of affine sorbent for purification of substance under study has been designed. The above instrument was used to choose the most-eligible of 38 sorbents with different dyes, which had gold-yellow dye ICI Procion Yellow 4 as the ligand. This sorbent made it possible to purify hemolysin II from B. cereus to homogeneity with high output for the first time in the recent 30 years (9). The instrument was awarded with gold medal at the International Specialized Exhibition “BIOTECH WORLD`2006”, Moscow International Conference “BIOTECHNOLOGY & MEDICINE”, March 14 – 17, 2006.
Introduction
Dye-coupled affine sorbents are prevalent in different areas of biochemistry, molecular biology and medicine (1, 2). "Procion” dyes are most widely used for affinity chromatography of proteins. This is quite understandable, since they are commonly used for fabric coloration in textile industry and, accordingly, winnowed by high affinity to proteins of wool, flax, silk, etc. [3, 4].
Immobilized dyes are used to purify nearly homogeneous proteins. For instance, interferon for therapeutic purpose is purified only by dye-coupling technologies. In this case, IgG-chromatography (on coupled immune sorbents) even cannot compete. The chromatography on dye-coupled gels is profitable for IgG clearance from albumin, as well as for separation of enzyme isoforms, functionally active from inactive enzymes, mutants from "wild type" enzymes, etc. [5].
"Ñibàñãîn Â1u" acts as a NAD-analogue [6], but CoA-dependent enzymes, hydrolases, RNA- and DNA-specific enzymes, complement, and interferon can bind with it [7]. Moreover, "Ðãîñion Red NOT 3Â" has a substantially high affinity to NADPH+-dependent dehydrogenases and kinases.
Two yellow dyes (III and IV), highly similar in structure, have a great difference in affinity to the enzymes [8].
The capacity of dye-coupled sorbents can be 10-100 times higher than that of affine sorbents with immobilized nucleotides or other ligands.
Results
The analytical instrument consists of 20 minicolumns (11 ´ 50 mm) filled up with different dye-coupled affinity sorbents. The columns are fixed in a common chamber. One of the columns, as nonspecific sorption control, contains Biogel A 1.5m (Bio-Rad) without a dye. The instrument permits concurrent chromatography in all columns.
Fig.1 (À) Demonstrated chemical structure of ligand choice sorbent for purification of Hemolysine II B. cereus1 (B) Purification of HlyII from bacterial culture supernatants. SDS–polyacrylamide gel (12%) of the samples from each purification step.
Table 1. Demonstrated output of pure Hemolysin II over 50%.
Fig. 1.
(A) The chemical structure of Procion yellow MX-R. Puri.cation of HlyII.
(B) Puri.cation of HlyII from the bacterial culture supernatants. SDS–polyacrylamide (12%) gel of samples from each puri.cation step. Lane 1, molecular weight markers; lane 2, the culture concentrate; lane 3, a fraction pool from the phosphocellulose P11 column; lane 4, a fraction pool from the pseudo-a.nity column; lane 5, puri.ed HlyII protein after gel-.ltration on Superose 12.
(C) Puri.cation of HlyII from cells extracts. SDS–polyacrylamide (12%) gel of samples from each puri.cation step. Lane 1, clari.ed lysate; lane 2, a fraction pool from CM cellulose column; lane 3, gel-.ltration on AcA44; lane 4, gel-.ltration on Superose 12; lane 5, the puri.ed HlyII protein (a marker). Twenty microliters from samples from each fraction was boiled with one equivalent of 2· SDS sample bu.er, and 10 ll was loaded onto the gel. HlyII migrates lower than the 45 kDa marker.
(D) The expression of His-tagged HlyII, BL21(pET29-HlyII-His). Coomassie-stained SDS–polyacrylamide (12%) gel of bacterial cell lysates before IPTG induction and 1, 2, and 3 h after induction. The induced HlyII migrates lower than the 45 kDa markers, lane 1. Cell pellets from 1 ml of medium before and after induction were boiled with SDS sample bu.er and loaded on the gel.
Table 1
Discussion
Commercial companies release sorbents with the dyes that have shown positive results in research laboratories. At the same time, research laboratories hold to their own methods using high-priced technologies.
Only in laboratories with no high-priced equipment and sorbents there was a direct necessity to contrive a cheap and efficient instrument and to offer it to all research and technological laboratories and companies! Preliminary cost of the instrument with 20 columns is app. 600 EURO, whereas one 5-ml column with blue agarose is about 60 USD.
Following the DIRECTING PROTOCOL attached to the instrument, anyone can choose a suitable sorbent in one day and book it in desired amount. It is planned to release two series of instruments A and B. Sorbents are marked with ordinal numbers from 1à to 19à and from 1b to 19b.
IN ONE DAY YOU CAN CHOOSE A SUITABLE SORBENT AND BOOK IT IN ANY AMOUNT
Please address applications with letter of intent to:
117292 Moscow, Kedrova str. 8, building 2, room 503
Institute of Biomedical Technologies WTU
Director, D.Sc. (Medicine), Professor
Belyaev Mikhail Victorovich
REFERENCES
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3. Ivanov V.B. // Active dyes in biology ., Nauka Eds, 1982.
4. Scopes R. K. Die-Ligands and multifunctional Absorbents: An Empirical Approach to Affiniti Chromatografy //Anal. Biochem. 1987.- V. 165.— P. 235—246 [Rev].
5. Osterman L.A.// Chromatography of proteins and nucleic acids. Moscow, Nauka Eds. 1985
6. Thompson S. T., Steliwaqen F. //Proc. Nat. Acad.-1975- V.72.-P. 669-672.
7. Low C. R., Small D. A. P., Alkinson A. /'//Inter. J. Biochem.- 1981- v. 13.-P. 33-40.
8. 8 Low C. R. et all //Anai. Biochem.—1980—V. 104.-P. 23-28. 1,8 t. zn.
9. Andreeva J.I., Nesterenko V.F. et.all. //Purification and cytotoxic properties of Bacillus cereus hemolysin II // Protein Expression and Purification- 2006, 47,186 -193