Unit #2: Biomolecules NOTES.4: INTRO TO BIOMOLECULES & LIPIDS.
Application of multichannel flow electrophoresis to separation of biomolecules: a survey
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Application of multichannel flow electrophoresis to separation of biomolecules: a survey Zheng Liu,* Jin Wang, Jian Luo, Fuxin Ding and Naiju Yuan Department of Chemical Engineering, Tsinghua University, Beijing 100084, People’s Republic of China Multichannel flow electrophoresis (MFE) is a newly developed method for continuous separation of biological products at a preparative scale, In this short survey, the application of MFE in the separation of proteins, enzymes and antibodies are overviewed. # 1998 John Wiley & Sons, Ltd. J. Mol. Recogn. 11, 149–150, 1998 Keywords: electrophoresis; preparative electrophoresis; multichannel flow electrophoresis; proteins; enzymes; antibodies Introduction Electric field is a powerful agent for the achievement of separation (Giddings, 1989). This is demonstrated by the excellent performance of electrophoresis techniques used for analytical purposes. However, the development of large- scale electrophoresis techniques is hindered by the difficul- ties in Joule heat dissipation, maintaining an expected pH gradient and preventing mixing of the products. Multichannel flow electrophoresis (MFE) is a newly developed technique for the separation of biomolecules based on their different isoelectric points (Liu et al., 1996a). Separation of charged species by MFE is carried out in a five-compartment electrolyser partitioned by membranes, in which the central compartment and the neighbouring compartments are used for sample loading and product discharging respectively. The electric field is applied perpendicular to the fluid flow in the compartments. The charged components are transmitted by the electric field from the central compartment into their corresponding elution compartments and washed out subsequently by carrier flow. The neutral component is carried through the central compartment. Various strategies are available for separation by MFE through different choices of buffering pH. For example, MFE can be operated at a pH that lies between the iso- electric points of two components for recovery of them simultaneously. MFE can also be carried out at the iso- electric point of a target component to remove the charged impurities out of the central compartment. One unique advantage of MFE is employing the elution compartments for product discharging. This makes it convenient to maintain a steady flow status inside the central compart- ment. Different from the conventional electrophoresis tech- niques, separation by MFE is conducted in an alternating electric field, whose negative part, running for seconds, can rapidly remove the concentration layers out of the surface of the membrane that separates the central and elution com- partments. Case studies have shown that the average migra- tion flux of BSA in a suitable alternating electric field was 40% higher than that in a steady one. No contamination of the products was found (Liu et al., 1996b). The unique advantages of this method also include high efficiency, ease of operation and mildness. Applying microfiltration membranes in MFE electrolyser construction increased the separation output of MFE to a level of 100 mg h 1 . Adding a hydrophilic polymer such as poly (vinyl alcohol) (PVA), poly (ethylene glycol) 4000 (PEG 4000) or poly (vinylpyroolidone) K 30 (PVP K 30) as shielding reagent in the sample solution effectively reduced the protein adsorption on the membrane during electro- phoresis. Consequently, the product fluxes were stable throughout the running period (Liu et al., 1997b). Separation of a Mixture of BSA and HBB The separation was buffered with 0.01 M, pH 6.0 Tris–HAc containing 250 ppm PEG 4000. The mixture solution contained 1.0 mg ml 1 each of BSA and HBB. In this work, two kinds of microfiltration membrane, HT Tuffryn and Supor provided by Gelmen Inc., were used to separate the central and elution compartments. In the case of HT Tuffryn the average product flux was 46.6 mg h 1 for BSA and 25.7 mg h 1 for HBB. In the case of Supor the output was 27.3 mg h 1 for BSA and 40.4 mg h 1 for HBB. Though the single-stage separation yield of continuous MFE was limited to 45%, the overall yield of MFE could be conveniently increased to a satisfactory level by recycling the residue from the central compartment for subsequent runs. Purification of Urokinase from Urine Extracts In the experiment, 0.02 M, pH 8.8 Tris–Gly buffer was used JOURNAL OF MOLECULAR RECOGNITION, VOL. 11, 149–150 (1998) # 1998 John Wiley & Sons, Ltd. CCC 0952–3499/98/010149–02 $17.50 * Correspondence to: Liu Zheng, Department of Chemical Engineering, Tsinghua University, Beijing 100084, People’s Republic of China. E-mail: [email protected]
Transcript of Application of multichannel flow electrophoresis to separation of biomolecules: a survey
Application of multichannel flow electrophoresis toseparation of biomolecules: a survey
Zheng Liu,* Jin Wang, Jian Luo, Fuxin Ding and Naiju YuanDepartment of Chemical Engineering, Tsinghua University, Beijing 100084, People’s Republic of China
Multichannel flow electrophoresis (MFE) is a newly developed method for continuous separation ofbiological products at a preparative scale, In this short survey, the application of MFE in the separation ofproteins, enzymes and antibodies are overviewed.# 1998 John Wiley & Sons, Ltd.
J. Mol. Recogn.11, 149–150, 1998
Keywords:electrophoresis; preparative electrophoresis; multichannel flow electrophoresis; proteins; enzymes;antibodies
Introduction
Electric field is a powerful agent for the achievement ofseparation (Giddings, 1989). This is demonstrated by theexcellent performance of electrophoresis techniques usedfor analytical purposes. However, the development of large-scale electrophoresis techniques is hindered by the difficul-ties in Joule heat dissipation, maintaining an expected pHgradient and preventing mixing of the products.
Multichannel flow electrophoresis (MFE) is a newlydeveloped technique for the separation of biomoleculesbased on their different isoelectric points (Liuet al.,1996a).Separation of charged species by MFE is carried out in afive-compartment electrolyser partitioned by membranes, inwhich the central compartment and the neighbouringcompartments are used for sample loading and productdischarging respectively. The electric field is appliedperpendicular to the fluid flow in the compartments. Thecharged components are transmitted by the electric fieldfrom the central compartment into their correspondingelution compartments and washed out subsequently bycarrier flow. The neutral component is carried through thecentral compartment.
Various strategies are available for separation by MFEthrough different choices of buffering pH. For example,MFE can be operated at a pH that lies between the iso-electric points of two components for recovery of themsimultaneously. MFE can also be carried out at the iso-electric point of a target component to remove the chargedimpurities out of the central compartment. One uniqueadvantage of MFE is employing the elution compartmentsfor product discharging. This makes it convenient tomaintain a steady flow status inside the central compart-ment.
Different from the conventional electrophoresis tech-niques, separation by MFE is conducted in an alternatingelectric field, whose negative part, running for seconds, canrapidly remove the concentration layers out of the surface of
the membrane that separates the central and elution com-partments. Case studies have shown that the average migra-tion flux of BSA in a suitable alternating electric field was40% higher than that in a steady one. No contamination ofthe products was found (Liuet al., 1996b). The uniqueadvantages of this method also include high efficiency, easeof operation and mildness.
Applying microfiltration membranes in MFE electrolyserconstruction increased the separation output of MFE to alevel of 100 mg hÿ1. Adding a hydrophilic polymer such aspoly (vinyl alcohol) (PVA), poly (ethylene glycol) 4000(PEG 4000) or poly (vinylpyroolidone) K 30 (PVP K 30) asshielding reagent in the sample solution effectively reducedthe protein adsorption on the membrane during electro-phoresis. Consequently, the product fluxes were stablethroughout the running period (Liuet al.,1997b).
Separation of a Mixture of BSA and HBB
The separation was buffered with 0.01 M, pH 6.0 Tris–HAccontaining 250 ppm PEG 4000. The mixture solutioncontained 1.0 mg mlÿ1 each of BSA and HBB. In thiswork, two kinds of microfiltration membrane, HT Tuffrynand Supor provided by Gelmen Inc., were used to separatethe central and elution compartments. In the case of HTTuffryn the average product flux was 46.6 mg hÿ1 for BSAand 25.7 mg hÿ1 for HBB. In the case of Supor the outputwas 27.3 mg hÿ1 for BSA and 40.4 mg hÿ1 for HBB.Though the single-stage separation yield of continuous MFEwas limited to 45%, the overall yield of MFE could beconveniently increased to a satisfactory level by recyclingthe residue from the central compartment for subsequentruns.
Purification of Urokinase from UrineExtracts
In the experiment, 0.02 M, pH 8.8 Tris–Gly buffer was used
JOURNAL OF MOLECULAR RECOGNITION, VOL.11, 149–150 (1998)
# 1998 John Wiley & Sons, Ltd. CCC 0952–3499/98/010149–02 $17.50
* Correspondence to: Liu Zheng, Department of Chemical Engineering,Tsinghua University, Beijing 100084, People’s Republic of China.E-mail: [email protected]
to preparesampleandcarriersolutions.Theflow ratesof thesampleand carrier for the centre, elution and electrodecompartments were 40, 160 and 320ml hÿ1 respectively.The applied electric field strengthwas maintained at 80V cmÿ1. In this casea gel membranesynthesizedaccordingto Yuan et al.’s (1993) patent was used to separatethecompartments.Continuousseparationfor 15min resultedinover 100000IU of urokinase product whose specificactivity was above40000IU mgÿ1. In comparison,puri-fication of urokinaseon thesamescalewasalsoconductedby ion exchange chromatography using CMC23, and theprocessing time was 72h besides the regenerationof theresin.Compared with this process,MFE hasexhibited itsdistinctive advantages suchashigh separation output,easeof operation and reagentsaving. Theseare of particularimportance whenlarge-scaleseparation is considered.
Purification of Anti-urokinase Mouse IgGCreated from Hybridoma Line
Theimmunizedmouseserumwasfirst precipitatedby 50%saturated(NH4)2SO4. Thentheprecipitate wasdissolvedin80ml of waterandsubjectedto dialysiswith waterandwiththe 0.02 M, pH 7.0 Tris–HAc, buffer usedfor continuousMFE. Hereagaina gel membranesynthesizedaccording toYuan et al.’s (1993) patent was used to separate thecompartments. Theelectrophoresiswascarried out undera60 V applied potential. The volume flow rates for thecentral, elution and electrode compartmentswere 40, 160
and360ml hÿ1 respectively.After thefirst run theproductwas adjusted to pH 7.0 and then placed in the centralcompartment for the second run, and analogously for thethird run.
SDS–PAGE analysis hasshown that no impurity bandsappeared in the column of IgG product. The molecularweights of thelight andheavychainsof IgG are24000and50000 respectively. A concentration test by the Bradfordmethod suggeststhat about 12.6mg of anti-urokinasemouse IgG was recovered from 15ml of mouseserum.The productivity of purifying mouse IgG wasabout2.1mghÿ1.
The conventional purification scheme of mouse IgGincludes ion exchange chromatography, gel elution chro-matography as well as dialysis procedures between eachstep, all of which aretime- andreagent-consuming andnoteasyto scaleup.Applying MFE to thepurification of mouseIgG presentsaneffectiveandeconomicwayof approachinga high separation output anda high resolution.
Concluding Remarks
The aboveresults havedemonstrated the workability andversatility of MFE in preparative-scale separation andpurification of biomolecules. Current work on MFE isfocusedonscalinguptheMFE apparatusandapplying MFEto theseparationof actual biomoleculemixtures,especiallythose with closerisoelectric points.
References
Giddings, J. C. (1989). Harnessing electrical forces for separation:capillary zone electrophoresis, isoelectric focusing, ®eld ¯owfractionation, split-¯ow thin-cell continuous separation andother techniques. J. Chromatogr. 480, 21±33.
Liu, Z., Huang, Z., Cong, J. Y., Ding, F. X. and Yuan, N. J. (1996a).Continuous separation of proteins by multichannel ¯owelectrophoresis. Sep. Sci. Technol. 31, 1427±1442.
Liu, Z., Yang, H., Huang, Z., Ding, F. X. and Yuan, N. J. (1996b).Multichannel ¯ow electrophoresis in an alternating electric®eld. Sep. Sci. Technol. 31, 2257±2271.
Liu, Z., Zhao, Y., Sheng, Z. -Y. Ding, F. X. and Yuan, N. J. (1997a).Hydrophilic polymer enhanced multichannel ¯ow electro-phoresis. Sep. Sci. Technol. 32, 1303±1313.
Liu, Z., Feng, S. H., Ding, F. X. and Yuan, N. J. (1997b).Multichannel ¯ow electrophoresis and its application inseparation and puri®cation of proteins, enzymes, and anti-bodies. Chinese J. Biotechnol. 12, 241±248.
Yuan, N. J., Liu, Z., Zhu, D. Q. and Ding, F. X. (1993). ChinesePatent Appl. 93-108630.2-4.
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# 1998JohnWiley & Sons,Ltd. JOURNAL OF MOLECULAR RECOGNITION,VOL. 11: 149–150(1998)
