A Novel Method for Diminishing Protein Aggregation during Denatuaration Process

Chinese Chemical Letters Vol. 17, No.3, pp 395-398, 2006395http://www.imm.ac.cn/journal/ccl.htmlA Novel Method for Diminishing Protein Aggregationduring Denatuaration ProcessYe Hua SHEN, Quan BAI, Yang Jun ZHANG, Yin Mao WEI,Hai Bo WANG, Xing Du GENG*Institute of Modern Separation Science, The Key Lab of Modern Separation Science in ShaanxiProvince, Northwest University, Xian 710069Abstract: The addition of packing material for high performance hydrophobic interactionchromatograghy (HPHIC) into the denaturant solution to prevent, or depress protein aggregationin the denatuaration process is presented. The renaturation of a-chymotrypsin (a-Chy)denatured with guanidine hydrochloride (GuHCI) solution indicated that renaturation efficiencycan be enhanced from 36. 1% to 59.0% by this new method. The structure of the ligand linkingof HPHIC packings is also important for the protein renaturation.Keywords: Protein renaturation, a-chymotrypsin, hydrophobic interaction chromatography.Many experimental results show that aggregation is the main side reaction in therenaturation process of proteins and once the proteins aggregate, it is rather difficult tobe renatured again. Therefore, the renaturation efficiency can be highly enhanced, ifthe aggregation is prevented or diminished. Many methods can partially prevent fromthe aggregation of denatured proteins, such as reverse micelles', chaperones', tetra-hydrofurfuryl alcohol (THFA)", polyethylene glycol (PEG)", surfactant，antibodies' andchromatography78 etc..Addition of high-perforamnce hydrophobic interactionchromatography (HPHIC) packing material into the denaturant solution of a.-chymo-trypsin (a-Chy) to prevent, or depress protein aggregation in the denatuaration processwas presented in this paper as a new method for an enhancement of renaturationefficiency.ExperimentalA Shimadzu LC- 10A system with LC- 10Atvp pumps and SPD- 10Avp UV-Vis detectormonitoring at 280 nm was used. The HPHIC packings was synthesized and packed inour lab. The mobile phases consisted of solutions A of 2.5 mol:L' (NH4)2SO4 +0.05mol:L" KH2PO4 (pH 7.0) and B of 0.05 mol-L' KH2PO4 (pH 7.0). The linear gradientwas run from 0-100%B in 25 min at flow rate of 1.0 mL :.min'5.0 mg a-Chy (Shanghai Reagent Company) was dissolved in 1.0 mL of 1.7E-mail: xdgeng@nwu.edu.cn中国煤化工MHCNMH G.396Ye Hua SHEN et al.mol:L"' guanidine hydrochloride (GuHC1) (marked as Gul.7) and 1.0 mL of 1.7 molL'GuHCl containing three kinds of HPHIC packing materials bound with ligands:tetrahydro- furfuryl alcohol (marked as K1.7), phenyl (marked as B1.7) and PEG600(marked as P1.7), respectively. The samples were then incubated at 25 C and stirredup at 200 rpm for 24 hours.In the same way, the silica gel (marked as Sil.7) and ligand substances, such astetrahydrofurfuryl alcohol were added into Gul.7 solution with the differentconcentrations, for the preparation of denatured a-Chy. Each sample of 200 μL wasinjected into the HPHIC column. The fractions, containing the refolded a-Chy whichhad the same retention time as native a-Chy, were collcted, and their bioactivityrecoveries were measured'.A specially designed injection unit, similar to a pre-column (shown in Figure1),was connected before the analytical HPHIC column.Take off the nut 2, thesuspension of 1.7 mol:L" GuHCl containing HPHIC packing material was added intothe pre-column from the side hole 2, then some mobile phase solution was added untilthe top level of the slurry reach nearly the bottle of the injection port. The nut 2 wastightly screwed on. Solution A flew into inlet 1 and outlet 3 to enter the analyticalcolumn (shown as C in Figure 2) for 10 min. The flow diagram of chromatographicseparation was shown in Figure 2.Figure 1 Scheme of injection columnFigure 2 Flow diagram_2BD↑0---0-、-≥3.|| F1. inlet for mobile phase; 2. port for injectingA. mobile phase; B. injection column;turbid solution; 3. outlet for mobile phaseC. analytical column; D. monitor; E. wasteResults and Discussiona-Chy was denatured with different concentrations of GuHCl. It was found when theconcentration of GuHCl was 1.7 mol:L' , the bioactivity recovery of a-Chy was 35.7%,which is very easy to compare the changes in the bioactivity recovery of a-Chy due tothe contributions of environmental factors to the renaturation of a-Chy. Therefore, thesolution 1.7 molL' GuHCl was selected as the denaturant concentration for a-Chy inthis study.0.20 mL of original samples (II), K1.7, B1.7 and P1.7 (the marks mentioned inExperimental), were taken, respectively, and centrifuged at 4C, at 5000 rpm for 10 min.The supermates (I) and the precipitated HPHIC packing (I) were put into thepre-column to renaturate a-Chy by HPHIC method. The advantages of the presentedmethod is that the original samples (II) can be renatured directly by HPHIC. A set ofchromato- grams of the separation of the denatured a-Chy with HPHIC were shown in中国煤化工YHCNMH G.A Novel Method for Diminishing Protein Aggregation397Figure 3 The chromatograms of the separation of the denatured a-Chy with HPHIC- ]_KI-.B1.7- _P1.7)2030t/minFigure 3. The chromatographic peaks were donated with the Arabinumerals. Thepeaks of the renatured a-Chy are marked with the symbol *”The bioactivityrecoveries of a-Chy were listed in Table1.From Table 1, the sum (I+I]) representing bioactivity recoveries of a-Chy isalmost the same as that of the original samples (II)I， The former (+1I) is slightlylower than the latter due to the centrifugation. The bioactivity recovery of a.-Chy inthe absence of HPHIC packings is less than 22.9% (from 36.1% to 59.0%). In thepresence of HPHIC packing, the bioactivity recovery of a-Chy is 1.6 folds of the former.The same result can be observed from Figure 3. The peak of the renatured a-Chy insample marked as K1.7 with HPHIC is highest. The result indicates that thebioactivity recoveries of a.-Chy can be enhanced to different degrees when thesehydrophobic packings were added in the denaturant solution.hydrophobic strength of these three packings is phenyl >THFA>PEG, whereas thecontributions of these packings to the renaturation efficiency were in the order ofTHFA> phenyl >PEG. Clearly, the structure of the ligand of hydrophobic packinggave the more contribution to renaturation than the hydrophobicity itself of thepacking*.The reason is that the secondary structure of proteins was destroyed in the GuHClsolution. When hydrophobic packings were present in the denaturant solution, theaggregation could be diminished due to the interaction of the hydrophobic groups ofhydrophobic packing with the protein intermediates. This interaction is very similar toTable 1 The bioactive recovery of a. Chy obtained from the different components in samplesR(%)*SamplesPrecipitated HPHICOriginalsupernates II+IIpackings IIsamples 1Gul.736.1K1.734.123.457.559.0B1.736.06.953.955.8P1.7.944.947.0*: R is the bioactivity recoveries of the renatured a-Chy by HPHIC.中国煤化工MHCNMH G.398Ye Hua SHEN et al.that of molecular chaperone with proteins'. Furthermore, the energy produced frominteraction between proteins and HPHIC packings can promote the denatured proteins tofold into natural conformation'Therefore, the renaturation efficiency can bsignificantly enhanced.In order to investigate the denatured mechanism in the presence of HPHIC packingmaterials, the contributions of naked silica and the free ligands, such as THFA tobioactivity recoveries of a-Chy were also studied. The results indicated that the nakedsilica gel neither can contribute to the renaturation of proteins nor enhance therenaturation efficiency, and when 3.2% THFA was added into the denaturing solution,the bioactivity recovery of a-Chy renatured with HPHIC can be increased from 36.1%to 50.3%, which is still lower than that in the presence of HPHIC packings with THFAligand (59.0%).In a short words, a novel method for an enhancement of renaturation efficiency byadding HIC packing material into the denaturant solution to prevent, or depress proteinaggregation in the denatuaration process is presented. The study on the renaturation ofa-Chy indicated that renaturation efficiency can be enhanced by this new method.AcknowlenentThese projects were supported by the National Natural Science Foundation of China (No.39880003 and 20175016).References1. A.J. Hagen, T. A. Hatton, D. I. C. Wang, et al., Biotech. Bioeng., 1990, 35(10), 955.N. J. Bulleid, R. B. Freedman, Nature, 1990, 335, 649.Y. H. Shen, H. B. Wang, Y. M. Wei, et al, Chin. J. Anal. Chem, 2002, 12(30), 1440.4. J. L. Cleland, S. E. Builder, J. R. Swartz, et al, BioTechnology, 1992, 10, 1013.J. D. Carlson, M. L Yarmush, Bio/Thchnology, 1992, 10, 86.6. J. Buchner, R. Rudolph, Bio/Techmology, 1991, 9, 157.7. X. D. Geng, X. Chang, J. Chromatogr, 1992, 599, 185.8. L. A. Guo, X. D. Geng, Chin. J. Biotechn, 2000, 16(6), 1). J. E. Folk, E. W. Schirmer, J. Biolog. Chem, 1965, 240(1), 181.l0. X. D. Geng, J. Zhang, Y. M. Wei, Chinese Science Bulletin, 2000, 45(3), 237.Received 24 August, 2005中国煤化工MHCNMH G.