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Anal. Chem. 1992, 64, 507-511
Determination of Recombinant Human Proinsulin Fusion Protein Produced in Escherichia coli Using Oxidative Sulf itolysis and Two-Dimensional HPLC Jeffrey
S.Patrick and Avinaah L.Lagu*
Lilly Research Laboratories, A Division of Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285
A method for the sample preparatlon and determlnatlonof a hunan prolnrulln fudon protdn (ChPI) expressed In recombinant €sche&h/a odlsampler Is dercrlbed. The method k applkable to samples contalnlng whole cells or Isolated Inclurkn bodlet. The procedure Invokes the rapld rumtolydr of samples In 7 M guanldlne hydrochloride and analyyrk wlth a coknn-swttch method udng dre exclwkn and weak adon exchange HPLC. The response of the method was linear for ChPI-$-sulfonate concmtratknr up to 4.4 mg/mL. Recovery of standard added to samples was greater than 85% In all c a w . The rp.clflclty of the method was demonstrated by the analyyrk of E . toll celb contalnlng a negatlve control plamld. The reproduclblllty of the method was good on a dally ( % RSD = 1.818; n = 18) and a day-today ( % RSD = 3.346; n = 28 days) bask. The general appllcablity of thk approach was suggested by quantltatlng recomblnant tryp rlnogen (methlonyltryplnogen expressed In E . -I), as the S-subnate, udng &e exdurkn and catlon exchange HPLC.
INTRODUCTION The production of heterologous proteins in Escherichia coli through recombinant DNA (rDNA) technology has been accomplished successfully in numerous cases.l-12 Many animal or human proteins have been expressed in E. coli as a fusion of the target protein and all or a portion of a host protein.1-3399*11 Fusion proteins are frequently designed to protect the protein of interest from p r o t e ~ l y s i s , ~to~ provide -~~ a specific method by which to isolate the fusion protein,16 or to provide a means of readily releasing the target protein from the expressed ~ r o t e i n . ' - ~ 8 , ~The J ~ Jisolation ~ and purification of these fusion proteins from the cells or the medium have been described briefly in studies of individual protein~'-~f'.~J~ and in reports describing isolation and purification procedures.lGm The production and isolation of these recombinant fusion proteins have been monitored by SDS-PAGE,1.2.4-9 Western blot? ELISA,l' bioassay,"l0 or HPLC of the fusion protein2lS2or the released target protein.lOJ1These methods are nonspecific,'$@ time consuming,'OJ1use harsh conditions (e.g. 60% formic acidla or are useful for only properly-folded, soluble target protein."" There have been examples of the use of capillary e l e c t r o p h ~ r e s i s ~and ~ - ~various ~ forms of HPLC3~5~7JoJ1~27-u to analyze isolated and purified proteins from rDNA sources. There have, however, been few reported studies which have dealt with the quantitative analysis of insoluble fusion proteins in cells or inclusion bodies.21p22 The analysis of heterologous proteins in recombinant hosts presents many challenges to the analytical chemist. When E. coli is the host organism the heterologous proteins are frequently produced in an insoluble, denatured form (called inclusion b o d i e ~ ) ' - ~ l which ~ " ~ ~ accumulates in the cytosol of *Author to whom all correspondence should be addressed.
the organism. In order to measure the total amount of target protein formed, the cells must be lysed and the inclusion bodies must be solubilized. Lysis of the cells can be accomplished through enzymatic or mechanical means or they can be permeabilized chemically through the use of sodium dodecylsulfate (SDS) or guanidinium salts.*38 Solubilization of the inclusion bodies requires the use of denaturants such as urea,SDS, and guanidinium chloride.'*% In the inclusion bodies the proteins can exist in many insoluble forms including covalent and noncovalent polymers.s38 To achieve a single molecular entity the intermolecular and intramolecular disulfide bonds must be disrupted and the protein unfolded. This has been accomplished through the use of denaturants in the presence of reducing and by reaction of the sample with sulfite and tetrathionate under denaturing conditions as described previ~usly.~This method, known as sulfitolysis, is rapid and is specific for disulfides.s Sulfitollpis is described by the reaction protein-S-S-protein
+ 23032-
-
2 x protein-S-S03-
and results in the formation of the cysteine-S-sulfonatederivative of the protein. The inclusion bodies also contain RNA, DNA, salts, lipids, and other compounds56in addition to the proteinaceous material. These materials have the potential for interference in analytical methods due to size, charge, or hydrophobicity. Several different methods have been used in the analysis of isolated rDNA-derived proteins. These include RPHPLC,21*29~30~32 anion33and cation%exchange HPLC, size exclusion HPLC,3O capillary e l e c t r o p h ~ r e s i s , hydrophobic ~~-~~ interaction chromatography,40and affinity HPLC.31 These methods have proven especially useful for soluble, hydrophilic proteins. For proteins of low solubility or high hydrophobicity, specific techniques such as the use of formic acidz have been employed. Chimeric human proinsulin represents one such protein. It is hydrophobic and not readily soluble as its Ssulfonate. We describe here a rapid method for the solubilization, derivatization, and HPLC of a chimeric form of human proinsulin produced in E. coli. The method is suitable for the quantitative measurement of chimeric human proinsulin in samples containing intact cells or isolated inclusion bodies. The chromatography uses a coupled, multicolumn system similar to those described by Kopaciewicz and RegnierS4l The potential versatility of the derivatization and column-switch method is demonstrated by analyzing recombinant trypsinogen (methionyl-trypsinogen) as the S-sulfonate in E. coli broth samples. EXPERIMENTAL SECTION Reagents. The chimeric human proinsulin-S-sulfonate used in these studies was received from Lilly Research Laboratories (Eli Lilly and Company, Indianapolis, IN). The urea and potassium tetrathionate were puriss grade from Fluka Chemie (Ronkonkoma, NY). Disodium ethylenediaminetetraaceticacid dihydrate (EDTA) and methanol were from EM Science (Gibbstown, NJ). Tris(hydroxymethy1)aminomethane (TRIS)
0003-2700/92/0364-0507$03.00/00 1992 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 5, MARCH 1, 1992
was biotechnology grade from Fisher Scientific (McGaw Park, IL). Trypsinogen (from bovine pancreas) and all other chemicals were from Sigma Chemical (St. Louis, MO) and were of reagent grade or better. Water purification was done using a Milli-Q Reagent Water System (Millipore, Millford, MA). The 7 M guanidine hydrochloride sulfitolysis reagent was prepared by dissolving 669 g of guanidine hydrochloride in a 1OOO-mLgraduated cylinder in sufficientwarm (EL 80 "C) purified water to make a 950-mL total volume. The mixture was stirred until dissolved and then cooled to room temperature. To this was added 12.6 g of sodium sulfite (anhydrous), 6.1 g of TRIS, 3.2 g of potassium tetrathionate (anhydrous),and 0.37 g of EDTA. The solution was stirred until all salts were dissolved, and then the pH was adjusted to 8.5-8.7 using 6 M hydrochloric acid. The volume was adjusted to lo00 mL with purified water. The reagent was stable at 5 OC for 5 days (data not shown). Chromatography. Chromatography was performed on a system equipped with two Gilson Model 305 pumps acting as the gradient system, a Gilson Model 303 pump as the auxiliary pump (all with 10-mL washable heads), a Gilson Model 231/401 autosampler with a thermostated sample rack maintained at 5 OC with PolyScience Model 9100 refrigerated circulating temperature controller (Polyscience, Division of Preston Industries, Inc., Niles, IL) and equipped with a 1-mL syringe and 50-pL injection loop, a Gilson Model 116 UV-visible variable-wavelengthdetector, a Gilson Model 805 Manometric module, a Gilson Model 811 Dynamic Mixer (Gilson Medical Electronics, Middleton, WI). A switching valve was custom-madeusing a Rheodyne Model 9010 PEEK injection valve (Rheodyne, Berkeley, CA) and custom electronics and pneumatics. This is similar to the Model 732 electronichigh-pressureswitchingvalve (Alcott Chromatography, Norcross, GA). The valve was switched using a contact relay from the autosampler. The TSK DEAEdPW (7.5 X 75 mm) column was obtained from Beckman (Fullerton, CA), the TSK SP-5PW (7.5 X 75 mm) column was obtained from Supelco (Bellefonte, PA),and the Zorbax GF-250 (10 X 250 mm) column was obtained from MacMod Analytical Inc. (Chadds Ford, PA). For the anion exchange (WAX) the 'A" (loading)mobile phase was 20 mM TRIS in 7 M urea adjusted to pH 7.0 with hydrochloric acid. The "B" (eluting) mobile phase was 20 mM TRIS and 0.5 M sodium sulfate in 7 M urea adjusted to pH 7.0 with hydrochloric acid. For the cation exchange (SCX) the 'A" mobile phase was 40 mM monobasic potassium phosphate in 7 M urea adjusted to pH 3.5 with concentratedphosphoric acid. The 'B" mobile phase was 0.5 M in sodium sulfate, 40 mM in monobasic potassium phosphate, and 7 M in urea with the pH adjusted to 3.5 with concentrated phosphoric acid. The "A" mobile phases were also used for the SEC on the GF-250 column using auxiliary pump. Both the ion exchange and size exclusion columns were operated at ambient temperature. The flow rate for the ion exchange columns was 1 mL/min, and the flow rate for size exclusion column was 0.5 mL/min. The effluent from the ion exchange column was monitored at 280 nm. The column switching was performed by injecting sample directly on to the size exclusion column at start. After the injection the size exclusion effluent was directed to waste for 12 rnin. The effluent from the size exclusion column was then loaded on to the ion exchange column from 12 to 20 min to insure that all of the chimeric proinsulinS-sulfonateor trypsinogen-S-sulfonatewas captured. At 20 min the analyticalgradient was started and the size exclusion effluent was directed to waste. A linear gradient (0-20% B in 20 min) was used for analysis. The composition was changed to 100% B for column washoff, and then the column was equilibrated at 0% B for 20 min. Procedure. E. coli cells were grown under conditionsdescribed by Williams, et ala2for proinsulin fusion protein but scaled up as necessary. Fermentation broth samples were prepared by mixing equal volumes of broth and methanol. An aliquot of broth suspension containing 2-30 mg of chimeric proinsulin or 0.4-4 mg of trypsinogen was dispensed into a 16- X 125-mm roundbottom polypropylene culture tube (Fisher Scienctific, McGaw Park, IL). The sample was centrifuged for 15 min at 3000g in a Beckman Model TJ-6 Centrifuge equipped with a TH-4 rotor (Beckman,Fullerton, CA). The supernatant was discarded, and the cell pellet was suspended in an appropriate volume of sulfitolysis reagent, depending upon the expected amount of ChPI.
~~~
Table I. Effect of Loading TimeonAnalyteRecov20000Da) where problems of solubility or recoveky might be encountered using traditional HPLC. The use of 7 M urea can keep the proteins soluble and thereby maximize sample recovery from the column and may provide a single denatured protein form for HPLC analysis. The denaturation of the analyte protein should not present a problem since the expresed proteins are themselves in a denatured form in the inclusion bodies. The results of
ANALYTICAL CHEMISTRY. VOL. 64. NO. 5, MARCH 1. 1882
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for ow studies and E. Kroeff for insightful discussion.
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