We explored the feasibility of attaching charged tails to a target protein, in this case β-galactosidase, for selective recovery. In this process, an ion-exchange membrane was used for selective binding and release of β-galactosidase with the attached purification fusions. Strength of binding and purity of eluate increased with increasing tail length. In addition, activity yield was improved with the implementation of an intermediate partial elution recycle procedure

Heng, Meng H.

Thiem, Erika A.

English

Name not available

Selective Recovery of β-Galactosidase With Charged Fusion Tails Using Ion-Exchange Membranes

University of Northern Iowa

The Journal of the Iowa Academy of Science: JIASVolume 101 | Number Article 101994Selective Recovery of β-Galactosidase WithCharged Fusion Tails Using Ion-ExchangeMembranesErika A. ThiemIowa State UniversityMeng H. HengIowa State UniverstiyCopyright © Copyright 1994 by the Iowa Academy of Science, Inc.Follow this and additional works at: http://scholarworks.uni.edu/jiasThis Research is brought to you for free and open access by UNI ScholarWorks. It has been accepted for inclusion in The Journal of the Iowa Academyof Science: JIAS by an authorized editor of UNI ScholarWorks. For more information, please contact scholarworks@uni.edu.Recommended CitationThiem, Erika A. and Heng, Meng H. (1994) "Selective Recovery of β-Galactosidase With Charged Fusion Tails Using Ion-ExchangeMembranes," The Journal of the Iowa Academy of Science: JIAS: Vol. 101: No. 2 , Article 10.Available at: http://scholarworks.uni.edu/jias/vol101/iss2/10]our. Iowa Acad. Sci. 101(2):66-69, 1994 Selective Recovery of 13-Galactosidase With Charged Fusion Tails Using Ion-Exchange Membranes ERIKA A. THIEM and MENG H. HENG Department of Chemical Engineering, 231 Sweeney Hall, Iowa State University, Ames, IA 50011 We explored the feasibility of attaching charged tails to a target protein, in this case (3-galactosidase, for selective recovery. In this process, an ion-exchange membrane was used for selective binding and release of (3-galactosidase with the attached purification fusions. Strength of binding and purity of eluate increased with increasing tail length. In addition, activity yield was improved with the imple-mentation of an intermediate partial elution recycle procedure. INDEX DESCRIPTORS: fusion proteins, purification fusions, protein purification, ion-exchange, (3-galactosidase The ability to generically manipulate proteins has made it possible to mass produce those of interest. This breakthrough has lead to many technological advancements in science and industry. However, prob-lems arise when genetically engineered proteins need to be separated and purified. The cost associated with separating and purifying pro-teins to the standard of industrial use is a major production cost. In fact, purification and recovery costs can account for as much as 80 per-cent of the total manufacturing cost in the large-scale production of recombinant protein products (1). Thus, without new downstream processing techniques, the abundance of genetically engineered pro-teins may lead to little profitability. In an effort to keep pace with modern technology, new procedures are being developed for downstream processing in the areas of separa-tion and purification. Ion-exchange is a technique which has been in use for many years. The bind and release process allows proteins with a certain ionic charge to be separated from others having the opposite charge or no charge. Recent developments in biotechnology utilize genetically engineered proteins in ion-exchange procedures, in partic-ular proteins with attached purification fusions. Purification fusions enhance selectivity of ion-exchange operations as well as facilitate dif-ficult separations. In designing fusion "rails", additional charges sup-plied by charged amino acids are used to provide the fusion with a distinctive charge which readily facilitates purification. The fusion protein is made by combining the DNA of the target protein with DNA coding for the fusion tail. The result is the expression of the fusion tail in the target protein. For this work, a microporous membrane was selected as the ion-exchange vehicle for application with the purification fusions. Previous research in our laboratory has taken advantage of negatively-charged fusion proteins in examining other ion-exchange separation techniques (2,3). Cationic polyelectrolyte precipitation and aqueous two-phase extraction are two separation methods in which increased selectivity in separation was seen with the purification fusions. The decision to explore ion-exchange membranes for selective separation was based on its low cost, ease of use, and scale-up potential. The membrane provided a uniform matrix that allowed rapid binding kinetics without problems of diffusion or flow problems common with packed bed or chromatographic separations. The membrane was used in studying a model protein separation system where 13-galacrosidase was recovered from cell extracts pre-BGCDl BGCD5 BGCDll ... Gin Lys I Gly Asp Pro Met Ala Tyr ... Gin Lys I Gly Asp Pro (Asp)4 Ser Tyr ... Gin Lys I Gly Asp Pro (Asp)10 Ser Tyr Fig. 1 Amino Acid Sequences for Purification Fusions. pared with fusion tails. Prior research (4) has shown success in the use of purification fusions to enhance selectivity using hollow fiber ion-exchange membranes (HFIEM). Over six fold enrichment was observed when the HFIEM was used for ion-exchange. Similar results were expected with the ion-exchange membrane cartridge. MATERIALS AND METHODS Fermentation and Extraction The fusion tails were produced from Escherichia coli strain Yl089-l with coding for 13-galactosidase. The fusion tails were attached at the carboxyl terminus of the 13-galactosidase and consisted of a series of amino acids with asparrates supplying the negative charge. Three purification fusions of various lengths; BGCDl, BGCD5 and BGCDl 1 had been prepared previously (5). BGCDl was used as a control and contained one additional asparrate due to the restriction sire. Four more aspartates were fused to BGCD5 and 10 more to BGCDl 1. Figure 1 shows the specific sequences for each purification fusion tail. From typical pK values for amino acids in proteins, net charges of the fusion tails were estimated to be -22.48, -37.72 and -60.57 for BGCDl, BGCD5 and BGCDl 1 respectively with the wild type having a net charge of-18.4. A cleavage site exists between the first aspartate in the chain and praline to allow for removal of the tail. Tyrosine allows for ultraviolet detection. The cells were grown in Luria-Bertani medium and fermented for 8 to 9 hours in an environmental shaker (37°C and 100 rpm). The cells were then harvested by centrifugation (4°C and 15000 g). After 10 minutes of centrifugation, the cells were washed with a neutral Tris-HCl buffer and recentrifuged. At this stage the cells were stored at -70°C until ready for use. The cell walls were ruptured by sonicarion (Heat Systems Wl85 Sonifier, Ultrasonics Inc., Plainview, NY) to extract the protein from inside the cell. Six cycles of 30 second sonification followed by a 30 second cooling period were performed in a cold salt water bath. Cell debris was removed by centrifugation (4°C and 23000 g) for 30 min-utes. The filtered supernatants were then adjusted to 10 units/ml by diluting them with a potassium phosphate buffer containing 2-mer-captoethanol and magnesium chloride, pH 5.7 (Sigma Chemical, St. Louis, MO). Membrane Ion-Exchange Operation The adjusted cell extracts were passed through a microporous, ion-exchange membrane enclosed in a polypropylene housing (Acti-Disk ™Cartridge, FMC Corp., Rockland, ME). A matrix of quater-nary amine came installed on the membrane to supply the fixed posi-tive charge. Prior to loading the cartridge the membrane was equili-brated by washing with 0.089M potassium phosphate buffer (0. lM ionic strength, pH 6). The wash was fed by a peristaltic pump at a flow rate of 2 ml/min for 10 minutes. Twelve milliliters of the cell SELECTED RECOVERY OF 13-GALACTOSIDASE 67 extract were prepared and a 2 ml sample of the feed was reserved. The remaining 10 ml were loaded onto the membrane, also at a rate of 2 ml/min. The apparatus assembly is pictured schematically in fig-ure 2. After all of the feed was loaded onto the membrane, it was washed with 20 ml of the potassium phosphate buffer previously mentioned. The wash cleansed the membrane of any loosely-bound protein. Peristaltic Pump Sample Reservoir Effluent (UV Anaylsis) Fig. 2. Apparatus assembly with FMC QUAT. Courtesy of FMC Corporation, Pine Brook, NJ. Step Gradient Elution Following the wash, the protein was removed from the membrane by a series of elutions. Buffer solutions were prepared by adding sodi-um chloride to the potassium phosphate wash buffer with ionic strengths ranging from 0.2 to 1.1 M. A step gradient elution was performed by injecting the buffers through the cartridge in 3 ml aliquots. By the time the 1.1 M solution was injected, nearly all of the protein was eluted. The individual effluents were analyzed for protein and activity content. Cartridge Storage The cartridge was sterilized by washing with a 25 ml sample each of deionized water and 70% methanol. The disk was dried by passing air through a syringe and stored at room temperature for reuse. Assays Protein content and activity assays were performed immediately afrer the step gradient elution. A dye-binding assay was performed to determine protein content and concentration. Coomassie Brilliant Blue G-250 stain (Bio-Rad, Richmond, CA) was used with a bovine serum albumin standard. Enzyme activity was monitored by a timed assay where o-nitrophenyl-f3-D-galactoside (ONPG), was hydrolyzed by the f3-galactosidase. One unit of activity is defined as the amount necessary to hydrolyze one micromole of ONPG to o-nitrophenol and galactose per minute at pH 7 .3 and 3 7°C. Recycle With Intermediate Partial Elution In later runs a recycle was implemented to increase the activity yield. The cartridge was prepared, loaded and washed as was done previously. At this point the membrane was eluted with 3 ml of the potassium phosphate salt solution via a syringe. The ionic strength of the elutant depended on the number of aspartates in the fusion tail. BGCDl was eluted with 0.2M solution, BGCD5 with 0.3M and 0.4M for BGCDl 1. The increasing concentration reflected the increase in binding strength with tail length as discovered in our ini-tial runs. With the membrane eluted, the feed was recycled and the cartridge was washed again with 20 ml of potassium phosphate buffer. The protein was then eluted through the step gradient proce-dure as before. The purpose behind the partial elution was to remove any unde-sired proteins and create additional space on the membrane surface for the successful binding of more f3-galactosidase. In addition, an accompanying reduction of undesired protein components in the final product would be favorable. RESULTS AND DISCUSSION The selectivity in this ion-exchange operation can occur either during the binding or release of the protein. Selectivity enhancement provided by the fusion tails was examined in both cases. Selective binding took place as a result of the intermediate partial elution. In this process the intermediate elution caused all loosely-bound proteins to be eluted from the membrane. In addition, unwanted, neutral and positively charged ions were rinsed through. Hence, the membrane was cleaned and cleared in preparation for the reapplication of the feed during the recycle. The activity recovered in the effluent increased significantly with the recycle procedure. For example, an initial run ofBGCDl resulted in an activity recovery of 39 U/ml. In contrast, when the BGCDl feed was recycled, the activity recovered rose to 55 U/ml. Figure 3 >. +-' ·5 ~120 "O c 6100 .0 c :::l 0 80 "O Q) ..._ ~ 60 0 u Q) c::: 40 -0 D Unbound Activity E2I Activity Recovered BGCD1 BGCD1 Recycled BGCDS BGCDS BGCD11 BGCD11 Recycled Recycled Fig. 3. Activity recovery with intermediate elution and recycle proce-dure. 68 JOUR. IOWA ACAD. SCI. 101(1994) shows the relationship between the increased activity and the inter-mediate elution procedure. The first column of each set shows the amount of activity recovered without the recycle procedure and the second column corresponds to the amount of recovered activity with the feed recycled. Some of the activity was never recovered and may have been lost due to inactivation or remained bound to the mem-brane during elution. Inactivation occurred when the enzyme was allowed to sit at room temperature for lengthy periods of time. In a measure to reduce the procedure time, the two, 20 ml buffer washes were performed using a syringe rather than the peristaltic pump. This step reduced the length of the recycle procedure and the assay results showed less missing activity. With the implementation of the intermediate partial elution recy-cle procedure, an increase in product yield was seen when compared to the single pass procedure. More active 13-galactosidase was recov-ered in the elutions than without the recycle. However, no increase in specific activity was seen for the recycled runs. Selective release was enhanced through the use of the step gradient elution. Each fusion showed a peak at the ionic strength Omax> required for eluting the fraction with the maximum specific activity. The Imax values remained quite reproducible throughout the bat-tery of runs. An expected correlation between the fusion tail length and the Imax peak was soon proven; with the increase of aspartates in the tail the ionic strength required to release the protein increased. This can be attributed to the additional charge the aspartates provide to the fusion tails. Although protein was eluted throughout the gradient steps, at Imax the majority of the protein was washed off. Specific activity and purification factor peaks coincided with the Imax peaks; however, the maximum total protein concentration for all three fusions was eluted at 0.25 M. For BGCDl and the wild type enzyme, the peak came at 0.3 M. BGCD5 showed a broader range spanning from 0.4 M to 0.5 M with an Imax at 0.45 M. Similarly, BGCDl 1 had a broad peak, its maximum was at 0.55 M. An Imax value of0.3 M for commercial 13-galactosidase shows that the purification fusion tails exhibit stronger binding. More information about the elution steps can be derived from figures 4, 5 and 6. The figures show the step gradient elution profiles for BGCDl, BGCD5 and BGCDll respectively. Protein concentration peaks can be seen at 0.25 M for all three tails. As seen in figure 4, the fraction with the maximum specific activity had a value of 350 U/mg. BGCD5 peaked at 400 U/ml and BGCDl at 700 U/ml as shown in figures 5 and 6. To understand the level of enrichment each effluent sample had obtained, the purification factor was calculated. The purification fac-tor a is defined as the total specific activity divided by the specific activity of the feed. The purification factor for BGCDl ranged from 2.5 to 5 throughout the tests with an average of 4.0. As with specific activity, a increased with tail length. BGCD5 and an a of 4.5 and BGCD 11 an average a of 7 .0. Figure 7 shows a step gradient elution profile overlay with each fusion tail being represented. The different specific activity peaks have a direct correlation with the purification factor. Just as BGCDl 1 has the highest specific activity of the three tails, it too has the highest a. Throughout the testing, BGCDl 1 showed superior binding capa-bilities far exceeding those seen for BGCDl and BGCD5. Its high specific activity brought upon by its longer tail led to a high purifica-tion factor. The difference in specific activity and purification factor between BGCD5 and BGCDl 1 was substantially greater than that between BGCDl and BGCD5. It was hypothesized that more charges on the fusion tail yield stronger binding traits. Because BGCDl 1 bound to the membrane much stronger than either BGCDl or BGCD5, 11 aspartates in the tail could approach an opti-mal number for selective binding. Continued research with even longer fusion tails would prove or disprove this hypothesis. 45 40 - A - Activity -o- Protein I ':----'> i p I 0... v' \ • 'ai \ I ,._ •• J. \ ·-··-··--· ......... \ r-·-··-··-a 0- -0- -0 0-' , / ' / b - -{) a.-· -a - ·-ti.· - ·tr· - .o.- · - · - · - · - o. o~.~~~--r~~~.,.-~~---,~~~~~~~...,....~~----o 0.2 0.4 0.6 0.8 1.2 Total Ionic Strength (M) 400 c; 300 ~ ,. ·~ u <( 200 \! :a <:> a. (/) 100 0 Fig. 4. Step gradient elution profile for BGCDl cell extracts treated using FMC QUAT. 40 /"". - .o. - Activity \ -o - Protein I -0- -0 er ' .. \ I I '°' ' al- ....... .J. 'o- ~--- ~.- - .. - .. - ... ...6· - ·A tr·-~---c: "tr . - .0. - - ~- - . - . - . - • -!::. 0-r-~~~,.--~---,.---,.~~~-,--~~~..,.--~~---,~~~-+ 0 0.2 0.4 0.6 0.8 1.2 Total Ionic Slrength (M) 500 c; 400 E 2. ,. 300 .2 u <( 2 200 13 100 "' a. (/) Fig. 5. Step gradient elution profile for BGCDS cell extracts treated using FMC QUAT. 40 35 \ \ .. \ ' -.o. - Aciviry -o- Protein ... _ ........... ~· ._ -:2.- -0 - 0... ... t:r. - A-.-,;.· - . ~ - . . - A-.-,;..-'::?°:_--:_--:_--:_--:~ ·5-t--~~~,.--~~---,....,.......,....~--,.-~---,.~..,.--...,....~-..,.~...,....~-+ 0 0.2 0.4 0.6 0.8 1.2 Total Ionic Strength (M) aoo 700 600 w 500 2. ,. 400 -~ u <( 300 _g u "' 200 a. (/) 100 0 Fig. 6. Step gradient elution profile for BGCDl 1 cell extracts treated using FMC QUAT. SELECTED RECOVERY OF B-GALACTOSIDASE 69 800 700 600 c; E 2. 500 ::-:~ 400 < " 0 300 " c. (/) 200 100 I 0 ' 0 0.1 0.2 I 0 -o- 8GCD1 -•- BGCDS -0· · 8GCD11 i /\ ;" ?. t i \ I : b \/ I \ 1\ : \ \ I _'p ' , b. . . _/ o--o--o-·-2·.:::..-..---=.~:----o.3 o'.4 o'.s o'.s o:7 o.s o.9 u Total Ionic Strength (M) Fig. 7. Specific activity peaks increase with longer tail length. The enzyme binds more strongly to the ion-exchange membrane with an increasing number of aspartates in the tail. CONCLUSIONS In this work it was demonstrated that purification fusion tails can be used to enhance separation and recovery on ion-exchange mem-branes. Selective enhancement increased with fusion tail length. BGCDl 1 bound to the membrane most strongly followed by BGCDS and BGCDl. The bind and release process brought seven-fold enrichment of BGCD 11 from cell extracts through step gradient elution. The product was recovered in an active state with a specific activity level comparable to commercial ~-galactosidase. The inter-mediate partial elution procedure increased activity recovery by near-ly 20%, however, no funher enrichment was witnessed. ACKNOWLEDGMENTS We are grateful to Dr. Charles Glatz for his guidance and helpful discussions in preparing this manuscript. This material is based upon work supported by the National Science Foundation under Grant No. BCS-9108583. REFERENCES 1. FULTON S.P., A.J. SHAHID!, N.F. GORDON, and A.B. AFEYAN. 1992. Large-Scale Processing and High-Throughout Perfusion Chrom-atography. Bio/Techno!. 10:635-639. 2. PARKER, D.E., C.E. GLATZ, C.F. FORD, S.M. GENDEL, I. SUOMI-NEN, and M.A. ROUGVIE. 1990. Recovery of a Charge-fusion Protein from Cell Extracts by Polyelectrolyte Precipitation. Biotechno!. Bioeng. 36:467-475. 3. LUTHER, J.R. and C.E. GLATZ. Nov. 1990. Enhanced Partitioning in Aqueous Two-phase Systems Using Genetically Engineered 13-Galacto-sidase. Poster presented at AIChE Annual Meeting, Chicago, IL. 4. HENG, M.H. and C.E. GLATZ. 1993. Charged Fusions for the Selective Recovery of 13-Galactosidase from Cell Extracts Using Hollow Fiber Ion-Exchange Membrane Adsorption. Biotechno!. Bioeng. 42:333-338. 5. ZHAO,]., C.F. FORD, C.E. GLATZ, MA. ROUGVIE, and S.M. GEN-DEL. 1990. Polyelectrolyte Precipitation of 13-Galactosidase Fusions Containing Polyaspartic Acid Tails.}. Biotechno!. 14:273-284. 

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Selective Recovery of β-Galactosidase With Charged Fusion Tails Using Ion-Exchange Membranes

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University of Northern Iowa