214
E/E(riopIiuresi~1992. /3. 214-219
R. BischoK, D. Koeckiin and C Roitsch
[13] Gianazza, E., Giacon, P., Sahlin, P. and Righetti, P. G., Electrophoresis 1985. 6, :i3-56. [14] Tonani. C. and Righetti, P. G., Elecrrophoresis 1991, / 2 , 1011-1021. [15] Righetti, P. G. and Tonani, C., Electrophoresis 1991, 12, 1021-1027. [16] Laemmli, Li. K., Nature 1970,227, 680-685. [I71 Ornstein, L., Ann. N . I.:Acad. Sci. 1964, 121,321-349. [18] Davis. B. J., Ann. N . Y. Acad. Sci. 1964, / 2 / , 404-427.
Rainer Bischoff Dominique Roecklin Carolyn Roitsch
'.
**,Protein
Unit, Strasbourg
phoresis 1986. 7, 128-133. [20] Mosher, R. A , , Bier, M. and Righetti, P. G.. Electrophoresis 1986, 7, 59-66. [211 Righetti, P. G., Immohilized pH Gradients: Theory and Methodology, Elsevier, Amsterdam 1990. [22] Rimpilainen, M . and Righetti, P. G., Electrophoresis 1985,6.419-422.
Analysis of recombinant proteins by isoelectric focusing in immobilized pH gradients Isoelectric focusing in immobilized pH gradients (IEF-IPG) was used to analyze three different recombinant proteins. Recombinant leech hirudin (65 amino acids, three disulfide bonds) expressed in Saccharomyces cerevisiae as a secreted protein and purified by anion-exchange and reversed-phase chromatography proved to be homogeneous with regard to its isoelectric point (pl). In addition, the theoretical p l , calculated on the basis of the primary structure, corresponded precisely to the measured p l o f 4.30. IEF-IPG was further employed to follow the stability of recombinant hirudin at pH 9, indicating that deamidation occurred under these conditions. A variant of recombinant human a,-antitrypsin (AAT) (389 amino acids, one cysteine residue) expressed in Escherichia coli and purified by anion-exchange, metal chelate and hydrophobic-interaction chromatography appeared to be homogeneous by polyacrylamide gel electrophoresis under reducing and denaturing conditions as well as by various high performance liquid chromatography methods. However, some heterogeneity was detected by IEF-IPG between pH 5-6. The measured plvalues of 5.43-5.58 were slightly lower than the calculated p l based on the primary structure (5.72). This indicated deamidations of Asn or Gln residues. A recombinant Schistosoma mansoni parasite antigen, p28 (210 amino acids, one cysteine residue) obtained after intracellular expression in Saccharomyces cerevisiae and affinity purification on glutathione agarose was analyzed by IEF-IPG in a pH 7.3-8.3 gradient. It appeared to be heterogeneous with regard to its p l , with the major component having a plof 7.81 compared to the calculated value of 7.17. N-Terminal amino acid sequencing as well as amino acid composition analysis were performed on the separated forms of p28 after electroblotting showing the feasibility of combining IEF-IPG with subsequent analytical methods to obtain structural information.
1 Introduction Within the last decade, progress in molecular biology fermentation of recombinant microorganisms and process technology as well as in protein chemistry has allowed the large-scale production of recombinant proteins and their
Correspondence: Dr. Rainer Bischoff,Transgbne S. A,, Protein Analytical Unit, 11, Rue de Molsheim, F-67082 Strasbourg Cedex, France Abbreviations: AAT. ai-antitrypsin; Bis, N,N'-methylenebisacrylamide; CAPS, 3-[cyclohexylamino]-l-propanesulfonic acid; HPLC, high performance liquid chromatography; IEF-IPG, isoelectric focusing in immobilized pH gradients: PAGE, polyacrylamide gel electrophoresis; PI, isoelectric point; PVDF, polyvinylidene difluoride; p28, antigen of Schistosoma rnansoni; rHVZ-Lys 47, recombinant hirudin variant 2 with a Lys residue in position 47: SDS, sodium dodecyl sulfate
0VCH Verlagsgesellwhaft
[I91 Bianchi-Bosisio,A., Righetti, P. G . , Egen,N. B. and Bier. M..Electro-
mbH, D-6940 Weinheim, 1992
further development into pharmaceutical products. While some of these proteins are already approved for therapeutic use in humans, numerous others are presently in clinical development and thus prone to enter the process of approval by the health care authorities in the near future. The production of a recombinant protein can be divided into three principal parts: (i) fermentation, (ii) purification and (iii) analysis, with each of them contributing significantly to the cost of the final product. Analytical characterization of a recombinant protein is a challenging task due to its complex structure. In the following, isoelectric focusing in immobilized pH gradients (IEF-IPG) of three recombinant proteins with varying degrees of structural complexity will be described to illustrate this point. As a first example, leech hirudin, a polypeptide with potent anticoagulant activity due to its pronounced affinity for hu0173-0835/92/0404-021~$3 50+ 2 i / 0
Electrophoresis 1992. 13. 214-219
man a-thrombin (inhibition constant below 1pM) [1,2] was analyzed by IPG. The properties of hirudin have attracted considerable interest with regard to its potential as a human therapeutic. Since the quantities of hirudin that can be extracted from the leech were far too small to conceive it as a source for production, recombinant DNA technology was employed to produce significantly larger amounts in heterologous expression systems [3-71. A production system for recombinant hirudin variant 2 with a Lys residue in position 47 (rHV2-Lys 47) in Saccharomyces cerevisiae has been developed for the isolation of large amounts of functional inhibitor [8,9].In the present work,IEF-IPG was employed to confirm the homogeneity of the purified molecule and to assess its stability under basic pH conditions.
lsoelectric focusing or recombinant proteins
215
2.2 Apparatus and software IEF-IPG was performed in a temperature-controlled, horizontal electrophoresis unit (Ultrophore, Pharmacia-LKB) using a 2297 Macrodrive 5 power supply (Pharmacia-LKB). N-Terminal amino acid sequencing was performed on a 470A gas-phase or a 477A pulsed-liquid-phase sequenator equipped with on-line HPLC analyzers and an integrator or a microcomputer (Applied Biosystems). Amino acid analysis was performed on a 420A amino acid analyzer equipped with an on-line high performance liquid chromatography (HPLC) analyzer and a microcomputer (Applied Biosystems) after hydrolysis with 6~ HCl in the gas phase for24 h at 110 “C (Pic0 Tag, Millipore-Waters, Bedford, MA, USA). Calculation of theoretical plvalues was based on the pKvalues for the ionizable side chain groups of Arg (12.1), Lys (lO.O), His (6.3), Tyr (lO.l), Cys (8.5), Asp (3.8), Glu (4.4) and the N- and C-terminus (8.0 and 3.1), respectively, using the DNASTAR software package (Madison, WI, USA).
As an example of a more complex protein, a variant of recombinant human a,-antitrypsin (AAT Ala357, Arg358)was analyzed by IPG. AAT Ala3”, Arg3” was previously shown to be a potent inhibitor of human plasma kallikrein [lo], a serine protease which is involved in blood pressure regulation as well as in controlling the host defense systems of 2.3 Methods coagulation and fibrinolysis. Recombinant DNA technology was employed to obtain functional AAT A1a3”, Arg358 2.3.1 IEF-IPG from Escherichia coli [ l l ] .IEF-IPG was subsequently used to assess the homogeneity of the isolated variant with reIEF-IPG gels (125 X 260 X 0.5 mm) were prepared as degard to its isoelectric point (pf). scribed by Gorg eta/. [ 141. Sample application wells (approximately5 pL) were placed at the cathodic site of the gel. To extend the methodology of IPG, an antigen from the paThe gradient was formed between pH 3.8-4.8 for rHV2-Lys rasite Schistosoma mansoni (referred to as p28) [12]was stu47,pH 5.0-6.0 f ~ r A A T A l a ~ ‘ ~ , A rand g ~ ~pH ’ , 7.3-8.3 forp28, died. Schistosomiasis is a chronic debilitating disease afusing a microgradient mixer (7.5 mL chamber volume). The fecting about 200 million people mainlyin tropical and subgels had a total monomer concentration of 6% acrylamide tropical countries. The development of a vaccine is thereand a crosslinking C of 4% Bis. Isoelectric focusing was perfore of great public and scientific interest. Recombinant formed at 10°C. Electrode solutions were 10 mM NaOH p28 was previously shown to be efficiently expressed in Sacand 10 mM glutamic acid. Electrophoresis was performed charomyces cerevisiae as an intracellular protein [13]. IEFfor60minat500Vand 15mAwiththepowerlimitset to20 IPG was used to analyze purified recombinant p28 and the W prior to sample application. Proteins were focused overseparated protein bands electroblotted to a polyvinylidene night at 2500 V and 5 mA with the power limit set to 5 W. difluoride (PVDF) membrane for further analyses. Protein bands were stained with Coomassie Brilliant Blue G-250, according to Neuhoff etal. [15].In the case of rHV2Lys 47, fixing of the protein was performed for 30 min with 50% w/v trichloroacetic acid in water and staining was done 2 Material and methods for 15 min with 0.1% Coomassie Brilliant Blue R-250 in methanol/water/acetic acid (100/100/20, v/v/v). Water 2.1 Material containing 5% v/v methanol and 7.5% v/v acetic acid was Immobilines ( p K of 3.6, 4.6, 6.2, 7.0, 8.5 and 9.3) were ob- used for destaining for 30 min. tained from Pharmacia-LKB Biotechnology (Bromma, Sweden). PIMarkers were glucose oxidase ( P I 4.15) and 2.3.2 SDS electrophoresis soybean trypsin inhibitor ( P I . 4.55; Pharmacia-LKB Biotechnology). The solutions were kept at 4 “C. Acrylamide, The upper gels for the second dimension were prepared acN3”’methylenebisacrylamide (Bis), tris(hydroxymethy1)a- cording to Laemmli [ 161 or Gyenes eta/. [ 171.The upper gel mino methane (Tris), glycine, ammonium persulfate system of Gyenes etal. uses 7.s0/oTand 5 S % C in 375 mM (APS), N,N,N’,N,-tetramethylethylenediamine (TEMED) Tris/HCl,pH 8.8,containing 0.1% w/v SDS.The separating and sodium dodecyl sulfate (SDS) were obtained from Bio- gel was identical in both cases and contained 15%T and Rad Laboratories (Richmond, CA, USA). SDS was twice re- 1.2VoC. These conditions have been adapted for recombincrylstallized from ethanol before use. Milli-Q purified ant p28 (calculated molecular mass: 23721 Da).The protein water was used throughout the experiments (Millipore, bands were cut out of the IPG gel (with the GelBond) and Bedford, MA, USA). All other reagents were of the highest equilibrated in 200 mM Tris/HCI, pH 8.8,5% w/v SDS and quality available. PVDF membranes, Immobilon-P or Pro10%glycerol for 15 min and air-dried on a strip of Parafilm. Blott, were obtained from Millipore or Applied Biosystems These gel slices were subsequently placed on top of the Inc. (Foster City, CA, USA), respectively. GelBond was ob- SDS gel (1.2-cm-wide application wells) which had been tained from Pharmacia-LKB. All glassware used was electrophoresed for 1h in the presence of 10 mM glutacleaned with chromosulfuric acid and washed with water thione (free cid in the reduced form) and held in place with followed by ethanol prior to use. All solutions were 0.22 pm 0.5% agarose in running buffer (60 mM Tris, 192 mM glyfiltered. cine, pH 8.3, and 0.1% SDS). Electrophoresis was per-
216
Flecrruphore>i.s 1992. 13, 214-219
R. Bi!icholl', D. Roecklin and C. Roitsch
formed at 16 mAfor2 h with 100 mM thioglycolate added to the running buffer. Protein bands were transferred to a PVDF membrane in 10 mM 3-[cyclohexylamino]-l-propane sulfonic acid (CAPS) buffer, pH 11, containing 10% v/v methanol for 1h at 400 m A after gel equilibration in this buffer for 15 rnin [18]. Protein bands on the PVDF membrane were stained with a solution of 0.1 Yo w/v Naphthol Blue in 45 O/o v/v methanol and 7% v/v acetic acid in water for 2 min and destained with water. 2.3.3 Electroblotting In order to transfer protein bands directly from the IEFIPG gel to a PVDF membrane, the gel was equilibrated in 10 mM CAPS, pH 11, containing 10% v/v methanol and 0.01% w/v SDS for 15 min.The polyacrylamide gel was subsequently detached from the supporting GelBond with a thin nylon string (fishing line) and placed in the horizontal transfer tank used for electroblotting while still on its plastic support. The GelBond was carefully removed from the gel after it had been well immersed in the transfer buffer. Electrotransfer to the PVDF membrane was performed for 1 h a t 400 mAin CAPS bufler(see above) omitting the SDS. Protein bands on the membrane were stained with Naphthol Blue as described above.
at Asn residues, showed that a single deamidation would result in a significantly reduced p/ value that should be easily detectable by IEF-IPG (Table 1).As expected, incubation at pH 9 led to the appearance of additional protein bands with more acidic p l values (Fig. 1B). After an incubation time of 4 days at 4"C, two major new bands with plvalues of 4.18 and 4.13 were observed (lane 8 in Fig. lB), indicating the presence of two predominant deamidation sites within rHV2-Lys 47. The two residues deamidating most rapidly under these forced conditions were later assigned to the two Asn-Gly sequences (Asn" and Asnj3) by amino acid sequencing and mass spectrometry, corroborating the susceptibility of these sites to deamidation (A. Tuong e t d a n d R. Bischoff eta/., manuscripts in preparation). Upon prolonged incubation of up to 12 days, further protein bands with reduced plvalues appeared, indicating additional deamidations (Fig. 1B). These results showed that purified rHV2Table 1. Theoretical p1 values of rHV2-Lys 47 and three of its potential deamidation uroducts Protein
Deamidation site
Calculated PI'')
rHv2-I.y~47 rHV2-Lys 47 rHV2-Lys 47 rHV2-Lys 47
None Ami3 Ami3
4.30 4.17 4.17 4.06
Asp Asp Asp AmT3 Asp +
+
+
+
a) DNASTAR. Madison, WI, USA
3 Results A
3.1 Recombinant hirudin A number of analytical methods including IEF-IPG have previously been employed to assess the homogeneity and structural integrity of rHV2-Lys 47 [9,19,20], giving no indication of any deviation from the expected amino acid sequence. IEF-IPG in a pH 3.8-4.8 gradient provided a measured p l of 4.30 that correlated well with the plvalue calculated on the basis of the primary structure of rHV2-Lys 47 (Fig. 1A and Table l).The p l o f rHV2-Lys 47 as determined by IEF-IPG was assessed by its relative migration to the p l markers shown in Fig. IA, assuming a linear pH gradient. Since rHV2-Lys 41 contains 7 Asn and 3 Gln residues within its primary structure, including two Asn-Gly sites that were shown to be particularly prone to deamidation [21-231, further studies were undertaken to investigate its stability at pH 9. Calculations of the theoretical isoelectric points of rHV2-Lys 47, containing one or two deamidations
4.55
C- 4.30
4.15 ._)
B
4.55 -
Q
4*31
4.21 / 4.1 8 4.13 \ 4-07 4-05 9 4.01
/
4.1 5 -
\
1
2
3
4
5
6
7
8
Figure 1. (A) IEF-IPG ofrecombinant hirudin (rHV2-Lys 47) between pH 3.8-4.8
after purification by anion-exchange and reversed-phase chromatography (reproduced with permission from [9]). (B) IEFIPG of recombinant hirudin (rHV2-Lys 47) between pH 3.8-4.8 after incubation in 0.1 ~Tris/HCI,pH9,at4"Cfor4,5,6,7, 10 and 12 days, respectively (lanes 8-3). Lane (1) p l standards; (2) and (9) rHV2~ y 47 s (purified).
Electruphoresrs 1992, 13, 214-219
lsoelectric Ibcusing of recombinant proteins
Lys 47, produced in a recombinant yeast strain, was devoid of deamidated forms, but that deamidation occurred upon prolonged incubation at pH 9.
3.2 Recombinant variants of AAT Analysis of purified AAT Ala3”, Arg3jxby IEF-IPG in pH 5-6 IPG showed some heterogeneity (Fig. 2). This heterogeneity was observed in six different production runs, representing six independent fermentation and purification procedures, indicating that similar modifications of the protein had occurred in each case, which the purification procedure was unable to resolve. As a comparison, the theoretical pls O ~ A A T A I ~ ~and ~ ’two , Apotentially ~ ~ ~ ~ ~deamidated forms were calculated based on their amino acid sequences (Table 2), giving additional credit to the possibility that deamidations had caused the observed p l shift. As Asn residues followed by Gly were shown to be particularly susceptible to deamidation [21-23],A~n”~ andAsn314might be two potential deamidation sites and the experimental approach outlined in the following paragraph will be applied to further analyze these different forms. Table2. Theoretical pivalues O ~ A A T A I ~ ~and ~ ’three , A ~ofits ~ ~poten~ ~ tial deamidation products Protein
Deamidation site
Calculated 01“)
None Asn116+Asp -t Asp Asp Asn3I4 Aso
5.72 5.61
~~
AAT Ala357,Arg3s8 ~ , AAT ~ a - ”Arg3j8 AAT Ala3”, Arg3” AAT Alals7, Arg3j8
5.61 5.50
+
-t
a) DNASTAR, Madison, WI, USA
3.3 Recombinant p28 Development of recombinant p28 into a human vaccine against schistosomiasis necessitates, amongst other studies, the careful characterization of the purified protein. When expressed in Saccharomyces cerevisiae as an intracellular protein and purified by ion-exchange and glutathione
5,9
217
agarose affinity chromatography, p28 appeared to be heterogeneous upon IEF-IPG between pH 7.3-8.3 (Fig. 3); it has a calculated p l of 7.17, based on its primary structure, while the major protein band in IEF-IPG had a p l of 7.81. Minor bands at pH 7.66 and 7.67, as well as at a lower pH value (pl7.60), showed that the product was heterogeneous (Fig. 3). Only minor protein bands were observed below pH 7.30 in an extended pH gradient (data not shown). In an attempt to structurally characterize protein bands separated on IEF-IPG by amino acid sequencing and amino acid composition analysis, SDS-polyacrylamide gel electrophoresis (PAGE) was used in conjunction with electroblotting onto PVDF.Two bands (p17.8 1; Fig. 3) were treated together and two different upper gel systems were evaluated. When using the system described by Laemmli [16], diffuse protein bands were obtained in the second dimension. The protein in these bands was nevertheless concentrated enough for N-terminal amino acid sequence analysis at a level of 49 pmol after electrotransfer to a PVDF membrane (Table 3). The sequence corresponded to the N-terminus of p28 as deduced from its cDNA (AGEXIKVIYFDG), but His in position 4 was not detectable, and the overall yield calculated from the amount deposited on the IEF-IPG gel (approximately 1.6 nmol) to the initial sequencing yield was fairly low (approximately 3 O/o). A better concentration effect in the second dimension was obtained when using the upper gel system of Gyenes etal. [17] with the modification described in Section 2.3.2.A clear N-terminal sequence of the doublet bands at p17.66 and 7.67 (Fig. 3) with an initial sequencing yield of25 pmol was obtained, also allowing the identification of His in position 4 (Table 3). Amino acid analysis showed that the doublet bands represented about 18% of the major band (pl7.81), which made it possible to calculate an overall recovery from the amount deposited on the IPG gel to the initial sequencing yield of approximately 9%. These data emphasized the importance of obtaining concentrated protein bands in the second dimension to increase the efficiency of amino acid sequencing by Edman degradation. It was also confirmed that the different protein bands that had been separated by IEF-IPG were related to p28 with identical N-termini.
-
5,75,6 5,8
5,4 5,35,2 5,l5,O 5,5
1
2
3
4
5
6
Figure 2. IEF-IPG of recombinant AAT Arg3”, obtained from six different fermentation and purification processes (lanes 1-6) using a gradient between pH 5-6 (reproduced with permission from [111).
2 18
Elmtrophorrsis 1992, 13, 214-219
K. HischofT. I). Koecklin and C. Koitsch
pH 8.3
PI 7.81
Figure3. IEF-IPG between pH 7.3-8.3 of recombinant p28 (1 nmol = 24 pg were deposited) obtained after purification by anion-exchange and glutathione-agarose chromatography. The major protein band (approximately810 pmol; ~ 1 7 . 8 1 and ) the two minor components (approximately 140 pmol; p l 7.66 and 7.67) as well as a weak protein band at p17.60 (approximately 50 pmol) were electroblotted to a PVDF membrane for further analyses.
PI 7-66/ 7-67 .01-7.60
pH 7.3 Table 3. Comparison of sequencing yields (in pmol) over 12 cycles of Edman degradation of recombinant ~28-derivedforms separated by IEF-IPG and transferred to PVDF membranes 2 3 4 5 6 7 8 9 101112 Cycle 1 Amino acid Band at p f 7.81 IIEF-IPG)d’ Band at p17.81 (IEF-IPG+SDS-PAGE)b’ Bands at pf 7.66/7.67 (IEF-IPG)d’
A 106 49 25
G 70 45 43
H I E 7 42 51 17 n.d.
E/E(riopIiuresi~1992. /3. 214-219
R. BischoK, D. Koeckiin and C Roitsch
[13] Gianazza, E., Giacon, P., Sahlin, P. and Righetti, P. G., Electrophoresis 1985. 6, :i3-56. [14] Tonani. C. and Righetti, P. G., Elecrrophoresis 1991, / 2 , 1011-1021. [15] Righetti, P. G. and Tonani, C., Electrophoresis 1991, 12, 1021-1027. [16] Laemmli, Li. K., Nature 1970,227, 680-685. [I71 Ornstein, L., Ann. N . I.:Acad. Sci. 1964, 121,321-349. [18] Davis. B. J., Ann. N . Y. Acad. Sci. 1964, / 2 / , 404-427.
Rainer Bischoff Dominique Roecklin Carolyn Roitsch
'.
**,Protein
Unit, Strasbourg
phoresis 1986. 7, 128-133. [20] Mosher, R. A , , Bier, M. and Righetti, P. G.. Electrophoresis 1986, 7, 59-66. [211 Righetti, P. G., Immohilized pH Gradients: Theory and Methodology, Elsevier, Amsterdam 1990. [22] Rimpilainen, M . and Righetti, P. G., Electrophoresis 1985,6.419-422.
Analysis of recombinant proteins by isoelectric focusing in immobilized pH gradients Isoelectric focusing in immobilized pH gradients (IEF-IPG) was used to analyze three different recombinant proteins. Recombinant leech hirudin (65 amino acids, three disulfide bonds) expressed in Saccharomyces cerevisiae as a secreted protein and purified by anion-exchange and reversed-phase chromatography proved to be homogeneous with regard to its isoelectric point (pl). In addition, the theoretical p l , calculated on the basis of the primary structure, corresponded precisely to the measured p l o f 4.30. IEF-IPG was further employed to follow the stability of recombinant hirudin at pH 9, indicating that deamidation occurred under these conditions. A variant of recombinant human a,-antitrypsin (AAT) (389 amino acids, one cysteine residue) expressed in Escherichia coli and purified by anion-exchange, metal chelate and hydrophobic-interaction chromatography appeared to be homogeneous by polyacrylamide gel electrophoresis under reducing and denaturing conditions as well as by various high performance liquid chromatography methods. However, some heterogeneity was detected by IEF-IPG between pH 5-6. The measured plvalues of 5.43-5.58 were slightly lower than the calculated p l based on the primary structure (5.72). This indicated deamidations of Asn or Gln residues. A recombinant Schistosoma mansoni parasite antigen, p28 (210 amino acids, one cysteine residue) obtained after intracellular expression in Saccharomyces cerevisiae and affinity purification on glutathione agarose was analyzed by IEF-IPG in a pH 7.3-8.3 gradient. It appeared to be heterogeneous with regard to its p l , with the major component having a plof 7.81 compared to the calculated value of 7.17. N-Terminal amino acid sequencing as well as amino acid composition analysis were performed on the separated forms of p28 after electroblotting showing the feasibility of combining IEF-IPG with subsequent analytical methods to obtain structural information.
1 Introduction Within the last decade, progress in molecular biology fermentation of recombinant microorganisms and process technology as well as in protein chemistry has allowed the large-scale production of recombinant proteins and their
Correspondence: Dr. Rainer Bischoff,Transgbne S. A,, Protein Analytical Unit, 11, Rue de Molsheim, F-67082 Strasbourg Cedex, France Abbreviations: AAT. ai-antitrypsin; Bis, N,N'-methylenebisacrylamide; CAPS, 3-[cyclohexylamino]-l-propanesulfonic acid; HPLC, high performance liquid chromatography; IEF-IPG, isoelectric focusing in immobilized pH gradients: PAGE, polyacrylamide gel electrophoresis; PI, isoelectric point; PVDF, polyvinylidene difluoride; p28, antigen of Schistosoma rnansoni; rHVZ-Lys 47, recombinant hirudin variant 2 with a Lys residue in position 47: SDS, sodium dodecyl sulfate
0VCH Verlagsgesellwhaft
[I91 Bianchi-Bosisio,A., Righetti, P. G . , Egen,N. B. and Bier. M..Electro-
mbH, D-6940 Weinheim, 1992
further development into pharmaceutical products. While some of these proteins are already approved for therapeutic use in humans, numerous others are presently in clinical development and thus prone to enter the process of approval by the health care authorities in the near future. The production of a recombinant protein can be divided into three principal parts: (i) fermentation, (ii) purification and (iii) analysis, with each of them contributing significantly to the cost of the final product. Analytical characterization of a recombinant protein is a challenging task due to its complex structure. In the following, isoelectric focusing in immobilized pH gradients (IEF-IPG) of three recombinant proteins with varying degrees of structural complexity will be described to illustrate this point. As a first example, leech hirudin, a polypeptide with potent anticoagulant activity due to its pronounced affinity for hu0173-0835/92/0404-021~$3 50+ 2 i / 0
Electrophoresis 1992. 13. 214-219
man a-thrombin (inhibition constant below 1pM) [1,2] was analyzed by IPG. The properties of hirudin have attracted considerable interest with regard to its potential as a human therapeutic. Since the quantities of hirudin that can be extracted from the leech were far too small to conceive it as a source for production, recombinant DNA technology was employed to produce significantly larger amounts in heterologous expression systems [3-71. A production system for recombinant hirudin variant 2 with a Lys residue in position 47 (rHV2-Lys 47) in Saccharomyces cerevisiae has been developed for the isolation of large amounts of functional inhibitor [8,9].In the present work,IEF-IPG was employed to confirm the homogeneity of the purified molecule and to assess its stability under basic pH conditions.
lsoelectric focusing or recombinant proteins
215
2.2 Apparatus and software IEF-IPG was performed in a temperature-controlled, horizontal electrophoresis unit (Ultrophore, Pharmacia-LKB) using a 2297 Macrodrive 5 power supply (Pharmacia-LKB). N-Terminal amino acid sequencing was performed on a 470A gas-phase or a 477A pulsed-liquid-phase sequenator equipped with on-line HPLC analyzers and an integrator or a microcomputer (Applied Biosystems). Amino acid analysis was performed on a 420A amino acid analyzer equipped with an on-line high performance liquid chromatography (HPLC) analyzer and a microcomputer (Applied Biosystems) after hydrolysis with 6~ HCl in the gas phase for24 h at 110 “C (Pic0 Tag, Millipore-Waters, Bedford, MA, USA). Calculation of theoretical plvalues was based on the pKvalues for the ionizable side chain groups of Arg (12.1), Lys (lO.O), His (6.3), Tyr (lO.l), Cys (8.5), Asp (3.8), Glu (4.4) and the N- and C-terminus (8.0 and 3.1), respectively, using the DNASTAR software package (Madison, WI, USA).
As an example of a more complex protein, a variant of recombinant human a,-antitrypsin (AAT Ala357, Arg358)was analyzed by IPG. AAT Ala3”, Arg3” was previously shown to be a potent inhibitor of human plasma kallikrein [lo], a serine protease which is involved in blood pressure regulation as well as in controlling the host defense systems of 2.3 Methods coagulation and fibrinolysis. Recombinant DNA technology was employed to obtain functional AAT A1a3”, Arg358 2.3.1 IEF-IPG from Escherichia coli [ l l ] .IEF-IPG was subsequently used to assess the homogeneity of the isolated variant with reIEF-IPG gels (125 X 260 X 0.5 mm) were prepared as degard to its isoelectric point (pf). scribed by Gorg eta/. [ 141. Sample application wells (approximately5 pL) were placed at the cathodic site of the gel. To extend the methodology of IPG, an antigen from the paThe gradient was formed between pH 3.8-4.8 for rHV2-Lys rasite Schistosoma mansoni (referred to as p28) [12]was stu47,pH 5.0-6.0 f ~ r A A T A l a ~ ‘ ~ , A rand g ~ ~pH ’ , 7.3-8.3 forp28, died. Schistosomiasis is a chronic debilitating disease afusing a microgradient mixer (7.5 mL chamber volume). The fecting about 200 million people mainlyin tropical and subgels had a total monomer concentration of 6% acrylamide tropical countries. The development of a vaccine is thereand a crosslinking C of 4% Bis. Isoelectric focusing was perfore of great public and scientific interest. Recombinant formed at 10°C. Electrode solutions were 10 mM NaOH p28 was previously shown to be efficiently expressed in Sacand 10 mM glutamic acid. Electrophoresis was performed charomyces cerevisiae as an intracellular protein [13]. IEFfor60minat500Vand 15mAwiththepowerlimitset to20 IPG was used to analyze purified recombinant p28 and the W prior to sample application. Proteins were focused overseparated protein bands electroblotted to a polyvinylidene night at 2500 V and 5 mA with the power limit set to 5 W. difluoride (PVDF) membrane for further analyses. Protein bands were stained with Coomassie Brilliant Blue G-250, according to Neuhoff etal. [15].In the case of rHV2Lys 47, fixing of the protein was performed for 30 min with 50% w/v trichloroacetic acid in water and staining was done 2 Material and methods for 15 min with 0.1% Coomassie Brilliant Blue R-250 in methanol/water/acetic acid (100/100/20, v/v/v). Water 2.1 Material containing 5% v/v methanol and 7.5% v/v acetic acid was Immobilines ( p K of 3.6, 4.6, 6.2, 7.0, 8.5 and 9.3) were ob- used for destaining for 30 min. tained from Pharmacia-LKB Biotechnology (Bromma, Sweden). PIMarkers were glucose oxidase ( P I 4.15) and 2.3.2 SDS electrophoresis soybean trypsin inhibitor ( P I . 4.55; Pharmacia-LKB Biotechnology). The solutions were kept at 4 “C. Acrylamide, The upper gels for the second dimension were prepared acN3”’methylenebisacrylamide (Bis), tris(hydroxymethy1)a- cording to Laemmli [ 161 or Gyenes eta/. [ 171.The upper gel mino methane (Tris), glycine, ammonium persulfate system of Gyenes etal. uses 7.s0/oTand 5 S % C in 375 mM (APS), N,N,N’,N,-tetramethylethylenediamine (TEMED) Tris/HCl,pH 8.8,containing 0.1% w/v SDS.The separating and sodium dodecyl sulfate (SDS) were obtained from Bio- gel was identical in both cases and contained 15%T and Rad Laboratories (Richmond, CA, USA). SDS was twice re- 1.2VoC. These conditions have been adapted for recombincrylstallized from ethanol before use. Milli-Q purified ant p28 (calculated molecular mass: 23721 Da).The protein water was used throughout the experiments (Millipore, bands were cut out of the IPG gel (with the GelBond) and Bedford, MA, USA). All other reagents were of the highest equilibrated in 200 mM Tris/HCI, pH 8.8,5% w/v SDS and quality available. PVDF membranes, Immobilon-P or Pro10%glycerol for 15 min and air-dried on a strip of Parafilm. Blott, were obtained from Millipore or Applied Biosystems These gel slices were subsequently placed on top of the Inc. (Foster City, CA, USA), respectively. GelBond was ob- SDS gel (1.2-cm-wide application wells) which had been tained from Pharmacia-LKB. All glassware used was electrophoresed for 1h in the presence of 10 mM glutacleaned with chromosulfuric acid and washed with water thione (free cid in the reduced form) and held in place with followed by ethanol prior to use. All solutions were 0.22 pm 0.5% agarose in running buffer (60 mM Tris, 192 mM glyfiltered. cine, pH 8.3, and 0.1% SDS). Electrophoresis was per-
216
Flecrruphore>i.s 1992. 13, 214-219
R. Bi!icholl', D. Roecklin and C. Roitsch
formed at 16 mAfor2 h with 100 mM thioglycolate added to the running buffer. Protein bands were transferred to a PVDF membrane in 10 mM 3-[cyclohexylamino]-l-propane sulfonic acid (CAPS) buffer, pH 11, containing 10% v/v methanol for 1h at 400 m A after gel equilibration in this buffer for 15 rnin [18]. Protein bands on the PVDF membrane were stained with a solution of 0.1 Yo w/v Naphthol Blue in 45 O/o v/v methanol and 7% v/v acetic acid in water for 2 min and destained with water. 2.3.3 Electroblotting In order to transfer protein bands directly from the IEFIPG gel to a PVDF membrane, the gel was equilibrated in 10 mM CAPS, pH 11, containing 10% v/v methanol and 0.01% w/v SDS for 15 min.The polyacrylamide gel was subsequently detached from the supporting GelBond with a thin nylon string (fishing line) and placed in the horizontal transfer tank used for electroblotting while still on its plastic support. The GelBond was carefully removed from the gel after it had been well immersed in the transfer buffer. Electrotransfer to the PVDF membrane was performed for 1 h a t 400 mAin CAPS bufler(see above) omitting the SDS. Protein bands on the membrane were stained with Naphthol Blue as described above.
at Asn residues, showed that a single deamidation would result in a significantly reduced p/ value that should be easily detectable by IEF-IPG (Table 1).As expected, incubation at pH 9 led to the appearance of additional protein bands with more acidic p l values (Fig. 1B). After an incubation time of 4 days at 4"C, two major new bands with plvalues of 4.18 and 4.13 were observed (lane 8 in Fig. lB), indicating the presence of two predominant deamidation sites within rHV2-Lys 47. The two residues deamidating most rapidly under these forced conditions were later assigned to the two Asn-Gly sequences (Asn" and Asnj3) by amino acid sequencing and mass spectrometry, corroborating the susceptibility of these sites to deamidation (A. Tuong e t d a n d R. Bischoff eta/., manuscripts in preparation). Upon prolonged incubation of up to 12 days, further protein bands with reduced plvalues appeared, indicating additional deamidations (Fig. 1B). These results showed that purified rHV2Table 1. Theoretical p1 values of rHV2-Lys 47 and three of its potential deamidation uroducts Protein
Deamidation site
Calculated PI'')
rHv2-I.y~47 rHV2-Lys 47 rHV2-Lys 47 rHV2-Lys 47
None Ami3 Ami3
4.30 4.17 4.17 4.06
Asp Asp Asp AmT3 Asp +
+
+
+
a) DNASTAR. Madison, WI, USA
3 Results A
3.1 Recombinant hirudin A number of analytical methods including IEF-IPG have previously been employed to assess the homogeneity and structural integrity of rHV2-Lys 47 [9,19,20], giving no indication of any deviation from the expected amino acid sequence. IEF-IPG in a pH 3.8-4.8 gradient provided a measured p l of 4.30 that correlated well with the plvalue calculated on the basis of the primary structure of rHV2-Lys 47 (Fig. 1A and Table l).The p l o f rHV2-Lys 47 as determined by IEF-IPG was assessed by its relative migration to the p l markers shown in Fig. IA, assuming a linear pH gradient. Since rHV2-Lys 41 contains 7 Asn and 3 Gln residues within its primary structure, including two Asn-Gly sites that were shown to be particularly prone to deamidation [21-231, further studies were undertaken to investigate its stability at pH 9. Calculations of the theoretical isoelectric points of rHV2-Lys 47, containing one or two deamidations
4.55
C- 4.30
4.15 ._)
B
4.55 -
Q
4*31
4.21 / 4.1 8 4.13 \ 4-07 4-05 9 4.01
/
4.1 5 -
\
1
2
3
4
5
6
7
8
Figure 1. (A) IEF-IPG ofrecombinant hirudin (rHV2-Lys 47) between pH 3.8-4.8
after purification by anion-exchange and reversed-phase chromatography (reproduced with permission from [9]). (B) IEFIPG of recombinant hirudin (rHV2-Lys 47) between pH 3.8-4.8 after incubation in 0.1 ~Tris/HCI,pH9,at4"Cfor4,5,6,7, 10 and 12 days, respectively (lanes 8-3). Lane (1) p l standards; (2) and (9) rHV2~ y 47 s (purified).
Electruphoresrs 1992, 13, 214-219
lsoelectric Ibcusing of recombinant proteins
Lys 47, produced in a recombinant yeast strain, was devoid of deamidated forms, but that deamidation occurred upon prolonged incubation at pH 9.
3.2 Recombinant variants of AAT Analysis of purified AAT Ala3”, Arg3jxby IEF-IPG in pH 5-6 IPG showed some heterogeneity (Fig. 2). This heterogeneity was observed in six different production runs, representing six independent fermentation and purification procedures, indicating that similar modifications of the protein had occurred in each case, which the purification procedure was unable to resolve. As a comparison, the theoretical pls O ~ A A T A I ~ ~and ~ ’two , Apotentially ~ ~ ~ ~ ~deamidated forms were calculated based on their amino acid sequences (Table 2), giving additional credit to the possibility that deamidations had caused the observed p l shift. As Asn residues followed by Gly were shown to be particularly susceptible to deamidation [21-23],A~n”~ andAsn314might be two potential deamidation sites and the experimental approach outlined in the following paragraph will be applied to further analyze these different forms. Table2. Theoretical pivalues O ~ A A T A I ~ ~and ~ ’three , A ~ofits ~ ~poten~ ~ tial deamidation products Protein
Deamidation site
Calculated 01“)
None Asn116+Asp -t Asp Asp Asn3I4 Aso
5.72 5.61
~~
AAT Ala357,Arg3s8 ~ , AAT ~ a - ”Arg3j8 AAT Ala3”, Arg3” AAT Alals7, Arg3j8
5.61 5.50
+
-t
a) DNASTAR, Madison, WI, USA
3.3 Recombinant p28 Development of recombinant p28 into a human vaccine against schistosomiasis necessitates, amongst other studies, the careful characterization of the purified protein. When expressed in Saccharomyces cerevisiae as an intracellular protein and purified by ion-exchange and glutathione
5,9
217
agarose affinity chromatography, p28 appeared to be heterogeneous upon IEF-IPG between pH 7.3-8.3 (Fig. 3); it has a calculated p l of 7.17, based on its primary structure, while the major protein band in IEF-IPG had a p l of 7.81. Minor bands at pH 7.66 and 7.67, as well as at a lower pH value (pl7.60), showed that the product was heterogeneous (Fig. 3). Only minor protein bands were observed below pH 7.30 in an extended pH gradient (data not shown). In an attempt to structurally characterize protein bands separated on IEF-IPG by amino acid sequencing and amino acid composition analysis, SDS-polyacrylamide gel electrophoresis (PAGE) was used in conjunction with electroblotting onto PVDF.Two bands (p17.8 1; Fig. 3) were treated together and two different upper gel systems were evaluated. When using the system described by Laemmli [16], diffuse protein bands were obtained in the second dimension. The protein in these bands was nevertheless concentrated enough for N-terminal amino acid sequence analysis at a level of 49 pmol after electrotransfer to a PVDF membrane (Table 3). The sequence corresponded to the N-terminus of p28 as deduced from its cDNA (AGEXIKVIYFDG), but His in position 4 was not detectable, and the overall yield calculated from the amount deposited on the IEF-IPG gel (approximately 1.6 nmol) to the initial sequencing yield was fairly low (approximately 3 O/o). A better concentration effect in the second dimension was obtained when using the upper gel system of Gyenes etal. [17] with the modification described in Section 2.3.2.A clear N-terminal sequence of the doublet bands at p17.66 and 7.67 (Fig. 3) with an initial sequencing yield of25 pmol was obtained, also allowing the identification of His in position 4 (Table 3). Amino acid analysis showed that the doublet bands represented about 18% of the major band (pl7.81), which made it possible to calculate an overall recovery from the amount deposited on the IPG gel to the initial sequencing yield of approximately 9%. These data emphasized the importance of obtaining concentrated protein bands in the second dimension to increase the efficiency of amino acid sequencing by Edman degradation. It was also confirmed that the different protein bands that had been separated by IEF-IPG were related to p28 with identical N-termini.
-
5,75,6 5,8
5,4 5,35,2 5,l5,O 5,5
1
2
3
4
5
6
Figure 2. IEF-IPG of recombinant AAT Arg3”, obtained from six different fermentation and purification processes (lanes 1-6) using a gradient between pH 5-6 (reproduced with permission from [111).
2 18
Elmtrophorrsis 1992, 13, 214-219
K. HischofT. I). Koecklin and C. Koitsch
pH 8.3
PI 7.81
Figure3. IEF-IPG between pH 7.3-8.3 of recombinant p28 (1 nmol = 24 pg were deposited) obtained after purification by anion-exchange and glutathione-agarose chromatography. The major protein band (approximately810 pmol; ~ 1 7 . 8 1 and ) the two minor components (approximately 140 pmol; p l 7.66 and 7.67) as well as a weak protein band at p17.60 (approximately 50 pmol) were electroblotted to a PVDF membrane for further analyses.
PI 7-66/ 7-67 .01-7.60
pH 7.3 Table 3. Comparison of sequencing yields (in pmol) over 12 cycles of Edman degradation of recombinant ~28-derivedforms separated by IEF-IPG and transferred to PVDF membranes 2 3 4 5 6 7 8 9 101112 Cycle 1 Amino acid Band at p f 7.81 IIEF-IPG)d’ Band at p17.81 (IEF-IPG+SDS-PAGE)b’ Bands at pf 7.66/7.67 (IEF-IPG)d’
A 106 49 25
G 70 45 43
H I E 7 42 51 17 n.d.
