Journal of Biotechnology 147 (2010) 6472

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Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec

Expression and purication of recombinant human 1 -proteinase inhibitor and its single amino acid substituted variants in Escherichia coli for enhanced stability and biological activity
Saurabh Agarwal, Shweta Jha, Indraneel Sanyal , D.V. Amla
Plant Transgenic Lab, National Botanical Research Institute (CSIR), P.O. Box 436, Rana Pratap Marg, Lucknow, U.P. 226 001, India

a r t i c l e

i n f o

a b s t r a c t
Human 1 -proteinase inhibitor (1 -PI) is the most abundant protease inhibitor found in the blood and expression of biologically active recombinant 1 -PI has great potential in therapeutic applications. We report here the expression of a synthetic 1 -PI gene and its variants in Escherichia coli. Modied 1 -PI gene and its single amino acid variants were cloned in pMAL-c2X vector, which allowed expression of recombinant protein(s) as a fusion of maltose-binding protein (MBP) with factor Xa protease recognition site between the fusion partners. The synthetic gene(s) were expressed in different E. coli strains and maximum expression of recombinant 1 -PI and variants up to 24% of total soluble protein (TSP) was achieved with engineered strain carrying extra copies of tRNAs for rare codons. Recombinant 1 -PI protein(s) were puried by amylose afnity chromatography with high homogeneity and overall yield of about 79 mg l1 of bacterial culture (5.2 g wet cell mass). E. coli expressed recombinant 1 -PI showed specic anti-elastase activity and appeared as a single band of 45.0 kDa on SDS-PAGE. Primary structure of puried protein and integrity of N-terminus has been veried by mass spectrometric analysis. Recombinant 1 -PI expressed in E. coli was fully intact having molecular mass similar to native unglycosylated protein puried from human plasma. Increased thermostability and specic activities of puried 1 -PI variant proteins conrmed the stabilizing effect of incorporated mutations. Our results demonstrate efcient expression and purication of stable and biologically active 1 -PI and its variants in E. coli for further therapeutic applications. 2010 Elsevier B.V. All rights reserved.

Article history: Received 26 May 2009 Received in revised form 12 March 2010 Accepted 17 March 2010

Keywords: Recombinant 1 -proteinase inhibitor MBP fusion protein Modied synthetic gene E. coli expression Thermostability Site-specic mutation

1. Introduction Human 1 -proteinase inhibitor (1 -PI), also known as alpha-1antitrypsin (AAT) is an archetype of the serine protease inhibitor family and a major constituent in human plasma. Its key physiological function in human and animals is inhibition of neutrophil elastase in lungs, thus protecting pulmonary tissues from damage (Blank and Brantley, 1994). A single amino acid mutation (Glu342Lys) in the mobile domain results in 1 -PI deciency and potentially lethal hereditary disease causing lung emphysema and liver disorders (Lomas, 2005). Intravenous augmentation of puried 1 -PI from human serum is the only clinical treatment presently available, which is in great demand, always in limited supply and associated with possibility of pathogen contamination

This research was supported by Council of Scientic and Industrial Research (CSIR), India under the Network Project CMM 0004. Corresponding author. Tel.: +91 522 2297954/2297955; fax: +91 522 2205836/2205839. E-mail address: i sanyal@rediffmail.com (I. Sanyal). 0168-1656/$ see front matter 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2010.03.008

(Heresi and Stoller, 2008). Over expression of recombinant human 1 -PI in diverse alternative host systems has been considered over the period, however, none of them could full the demand of clinically safe and biologically active form of protein for therapeutic applications (Karnaukhova et al., 2006). The tertiary structure of 1 -PI share a common structure with other serpins and composed of three -sheets (A, B, C) and nine -helices (hAhI) as shown in Fig. 1A. In the native strained (S) active conformation, the molecule is intact and the reactive centre loop (RCL) is exposed to proteolytic cleavage. The cleavage accompanies an irreversible transition to a very stable relaxed (R) form where the newly created N-terminal portion of the cleaved loop is completely inserted as central strand of sheet-A, with concomitant loss of inhibitory activity (Whisstock et al., 2000). The shutter and the breach are two important areas for this conformational change (S R transition). The breach, located at the top of sheet-A, is the region where the RCL rst inserts. The shutter region is located in the middle of the serpin and controls the opening of the sheet-A. Both regions contain a number of highly conserved residues and several positions at which mutations result in hyperstability, without affecting inhibitory activity (Lee et al., 1996).

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Fig. 1. Tertiary structure of human 1 -PI protein, site-specic mutations in modied gene and chimeric gene construct. (A) Ribbon model of human 1 -PI protein molecule (PDB ID 1hp7) highlighting RCL, other critical domains such as breach and shutter domain and positions of incorporated single amino acid substitutions (encircled) to generate ve variants for increased stability and biological activity in recombinant 1 -PI. (B) Table showing ve incorporated point mutations and corresponding codon replacements in the modied synthetic 1 -PI gene and plasmid vectors. (C) Expression vector pMPI harbouring the modied synthetic 1 -PI coding sequence cloned in vector pMAL-c2X, downstream to the malE gene encoding for MBP fusion tag under the control of IPTG inducible ptac promoter and lacZ terminator with an in-frame cleavage site of protease factor Xa between MBP and 1 -PI fusion partners. Similarly ve independent expression vectors for 1 -PI variants were also generated with specic point mutations shown with arrows and designated as (I) pMFC, (II) pMFL, (III) pMAG, (IV) pMMV and (V) pMMI respectively.

Escherichia coli is often the host of choice for its ability to multiply rapidly to high cell densities on inexpensive media, availability of versatile vectors and achieving expression levels exceeding more than 30% of total cellular protein (Makrides, 1996; Baneyx, 1999). However, problems arise with expression of several heterologous genes for yield, stability and solubility of foreign proteins in E. coli due to factors including, presence of rare codons, translational efciency, stability of mRNA, insolubility, formation of inclusion bodies and complex post-translational modications (Makrides, 1996; Kane, 1995). Several strategies have been used to circumvent the codon bias, increase yields and stability of foreign proteins in E. coli such as exchange of rare codons with E. coli preferred codons, co-expression of desired tRNA genes and use of fusion proteins (Baneyx, 1999). Different fusion tags seem to improve solubility, stability, translational efciency and folding by acting as molecular chaperones in context to the partner proteins besides facilitating rapid purication (Esposito and Chatterjee, 2006; Kapust and Waugh, 1999). We have designed and developed a highly modied synthetic 1 -PI gene and its ve single amino acid substitution variants by extensive codon-optimization for dicot plant genes to achieve safe, stable and biologically active recombinant 1 -PI protein in transgenic plants (Agarwal et al., 2008). The designed gene contains 69 codons out of 394 (17%), which are reported to be rarely used in E. coli and their presence inhibits expression (Makrides, 1996). In the present study, the modied 1 -PI gene and its single amino acid substituted variants were efciently expressed under optimized culture conditions as MBP fusion protein, driven by isopropyl--d-thiogalactopyranoside (IPTG) inducible ptac promoter by overcoming the limitations

of codon bias, insolubility, misfolding, degradation or aggregation. The expressed protein(s) were puried by amylose afnity chromatography and characterized for its integrity, stability and biological activity. 2. Materials and methods 2.1. Construction of E. coli expression vectors with modied 1 -PI gene The coding sequence of human 1 -PI gene was codon-optimized according to the codon usage frequencies of highly expressed dicot plant genes and synthesized by PCR-based gene synthesis using overlapping oligonucleotides (Agarwal et al., 2008; Stemmer et al., 1995). Similarly, ve variants of modied 1 -PI gene for single amino acid substitutions at Phe51 to Cys (FC), Phe51 to Leu (FL), Ala70 to Gly (AG), Met358 to Val (MV) and Met374 to Ile (MI) have been developed by site-directed mutagenesis of respective codons as shown in Fig. 1B (An et al., 2005). These positions were selected for substitution as they were reported to be present at critical sites of the 1 -PI protein molecule (Im et al., 2004). Synthetic 1 -PI gene and its variants were amplied using forward primer 5 CGGAATTCggatccATGGAAGATCCTCAAGGAGATGCTGC-3 and reverse primer 5 -GGTACCTCTAGaagcttTTACTACTTCTGAGTAGGGTTAACC-3 to incorporate initiation and stop codons (underlined) and BamHI and HindIII restriction sites (lower case) at 5 and 3 end of the gene respectively. The amplied PCR products were cloned into E. coli expression vector pMAL-c2X (NEB, USA), downstream to the malE gene of MBP to develop vectors pMPI with modied native 1 -PI gene and pMFC, pMFL, pMAG, pMMV and pMMI with

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gene variants, using the standard molecular cloning techniques (Sambrook and Russel, 2001). 2.2. Expression and purication of recombinant 1 -PI from E. coli E. coli strains DH5, TB1 (NEB, USA) and BL21-CodonPlus-RIL (BL21CP) carrying tRNA genes for codon AGG/AGA, ATA and CTA (Stratagene, USA) were used as expression hosts. Recombinant E. coli strains were grown in LB medium with 100 g ml1 ampicillin at 37 C up to OD600 0.5, 1.0 or 2.0. The cultures were induced with 0.3 mM IPTG and further grown at either 37 C or 30 C, for 18 h. Aliquots of induced cultures were withdrawn at periodic intervals, harvested by centrifugation (4000 g for 10 min at 4 C) followed by washing and resuspension in extraction buffer (EB; 20 mM TrisHCl, pH 7.4, 200 mM NaCl, 1 mM EDTA, 1 mM sodium azide, 10 mM -mercaptoethanol). Cells were lysed by sonication with short pulses and debris was removed by centrifugation (9000 g for 20 min at 4 C). The crude extract from 1 l E. coli culture was diluted and applied on a pre-equilibrated amylose column (5 ml, NEB, USA) followed by washing with 12 column volumes of EB and elution of bound proteins with EB supplemented with 10 mM maltose. The fractions containing fusion protein were pooled and concentrated followed by cleavage with protease factor Xa in cleavage buffer (20 mM TrisHCl, pH 8.0, 100 mM NaCl, 2 mM CaCl2 ) to separate MBP and 1 -PI fusion partners. The cleaved reaction products were again applied on second amylose column followed by collection of pure 1 -PI protein in ow-through fractions. Puried protein was analyzed by SDS-PAGE, Western immunoblotting, DAC-ELISA and residual PPE activity assay. TSP was determined by dye binding procedure taking bovine serum albumin as a reference standard (Bradford, 1976). 2.3. SDS-PAGE and Western immunoblotting Protein samples combined with Laemmli buffer were boiled for 10 min and fractionated on 10% SDS-PAGE gel, followed by staining with 0.1% Coomassie brilliant blue R-250 (Sambrook and Russel, 2001; Laemmli, 1970). Electrophoresed protein samples were transferred onto polyvinylidene diuoride (PVDF) membrane (Bio-Rad, USA) in transfer buffer (25 mM Tris base, 192 mM glycine, pH 8.3, 0.1% SDS) for Western immunoblotting. The membranes were blocked in 5% non-fat dry milk (in PBST) at 25 C followed by incubation with either rabbit anti-human 1 -PI antibody or rabbit anti-MBP antibody. The membranes were washed and incubated with goat anti-rabbit IgG-alkaline phosphatase conjugated antibody followed by colour development with BCIP-NBT substrate solution. 2.4. Quantitative estimation of recombinant 1 -PI by DAC-ELISA Recombinant 1 -PI protein was quantied by DAC-ELISA method (Agarwal et al., 2008). Aliquots of protein samples were coated in wells of microtiter plate followed by blocking and incubation with 1:5000 dilution of rabbit anti-human 1 -PI antibody, washing with PBST and incubation with 1:8000 dilution of goat anti-rabbit IgG-alkaline phosphatase conjugated antibody followed by colour development with p-nitrophenyl phosphate substrate solution. Expression levels of recombinant 1 -PI were quantied on a linear standard curve plotted with pure human 1 -PI protein (Sigma, USA). 2.5. Residual PPE activity assay and thermostability analysis The biological activity of recombinant 1 -PI in cell free extracts and puried samples was determined as residual porcine

pancreatic elastase (PPE) activity using N-succinyl-Ala-Ala-Alap-nitroanilide as chromogenic substrate (Agarwal et al., 2008). Protein samples (50 l) were added into 100 l of assay buffer (20 mM TrisHCl, pH 8.0, 150 mM NaCl, 0.01% Tween-80) followed by addition of 50 l (3.0 g ml1 ) PPE, incubation at 37 C for 15 min and addition of 50 l of 2 mM chromogenic substrate and further incubation for 2 h at 25 C. The activity of residual PPE was determined by measuring the release of p-nitroaniline from chromogenic substrate at 405 nm. Pure human 1 -PI protein was used as reference standard for corresponding recombinant 1 -PI concentration and residual PPE activity. Specic activity was based on amount of biologically active 1 -PI per mg of total soluble protein. The stability of wild type and mutant recombinant 1 -PI proteins expressed in E. coli was determined as percentage of remaining biological activity at 37 C and 54 C and compared with native glycosylated human serum 1 -PI. The aliquots (5 g) of puried protein samples (50 g ml1 ) were incubated at 37 C and 54 C for 2.5 h and 1 h, respectively. Aliquots were withdrawn after interval of every 30 min and 10 min at 37 C and 54 C respectively and analyzed for the remaining 1 -PI activity by residual PPE enzymatic activity assay. All the assays were performed in triplicates along with internal control. 2.6. Mass spectrometric analysis Coomassie brilliant blue stained protein bands of interest were cut out from the SDS-PAGE gel and in-gel digested as described by Shevchenko et al. (2006). Proteins were reduced with dithiothreitol and alkylated with iodoacetamide before overnight digestion with 0.02 g l1 trypsin (Proteomics grade, Sigma, USA) at 37 C. The recovered peptides and intact protein samples were desalted with C18 and C4 Zip-Tip, respectively according to the manufacturers instructions (Millipore, USA). Samples were prepared by the dried droplet method using -cyano-4-hydroxycinnamic acid (CHCA) as matrix dissolved in 50% (v/v) acetonitrile and 0.1% (v/v) triuoroacetic acid. The MS and MS/MS spectra were acquired using 4800 MALDITOF/TOF mass spectrometer (Applied Biosystems, USA) equipped with a Nd:YAG (355 nm, 200 Hz) laser. The instrument was operated with delayed extraction (1100 ns) at accelerating voltage of 20 kV. The 4700 cal mix (having mixture of six standard peptides) and bovine serum albumin (Applied Biosystems, USA) were used as external calibrants for reector and linear mode, respectively. The MS and MS/MS spectra were typically acquired by averaging 30 sub-spectra from a total of 900 shots of the laser with the intensity set at 4200 and 4900, respectively. Protein identication from the generated data was performed by searching against the MSDB database using online Mascot search engine (http://www.matrixscience.com/). Peptide mass tolerance of 100 ppm and fragment ion mass tolerance of 0.2 Da was set and maximum missed cleavages allowed was one. Carbamidomethylation (C) as xed modication, deamidation (NQ) and oxidation (M) as variable modications were considered. The probability score calculated at p < 0.05 was used as the criteria for correct identication of proteins. 3. Results 3.1. Synthesis of modied 1 -PI gene and recombinant vectors The coding sequence of human 1 -PI gene was designed to display codon usage pattern of abundantly expressed dicot plant genes. In the modied 1 -PI gene (GenBank accession no. EF638826), 205 codons out of 394 (52%) were replaced with substitution of 281 favoured nucleotides. Codon frequency analysis revealed that modied 1 -PI gene contains 69 codons (17%), which

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Fig. 2. Expression of MBP1 -PI fusion protein in E. coli under different culture conditions. (A) Growth pattern of recombinant bacterial strains DH5 ( ), TB1 () and BL21CP ( ) with pMPI expression vector at 37 C after induction with IPTG at OD600 0.5 (arrow). (B) Comparative yield of expressed fusion protein in E. coli BL21CP with IPTG induction at 25 C ( ), 30 C ( ) and 37 C (). (C) Yield of fusion protein in different strains of E. coli BL21CP ( ), TB1 () and DH5 ( ) at 30 C after IPTG induction. (D) SDS-PAGE analysis of cell-free extracts of recombinant BL21CPpMPI culture induced for different time periods. Lane 1, prestained molecular mass standard; lane 2, recombinant BL21CP having pMAL-c2X vector without 1 -PI modied gene; lane 310, recombinant BL21CP transformed with pMPI expression vector induced for 18 h respectively at 30 C with expression of 87.5 kDa fusion protein as indicated by arrow.

were reported to be rare for E. coli and among them 19 (5%) are most rarely used codons (AGA/AGG, ATA, CTA, CCA and CCC). To express recombinant 1 -PI and its variants in E. coli, the modied 1 -PI coding sequence(s) were inserted downstream of the malE gene encoding MBP fusion tag in the bacterial expression vector pMAL-c2X under the control of IPTG inducible ptac promoter. Six expression vectors such as pMPI, pMFC, pMFL, pMAG, pMMV and pMMI containing in-frame cleavage site of factor Xa protease between MBP1 -PI fusion partners were developed (Fig. 1C). 3.2. Expression of recombinant 1 -PI in E. coli In order to optimize the host strain and conditions for maximum induction, the recombinant E. coli strains DH5, TB1 and BL21CP transformed with pMPI expression vector were grown and induced separately by addition of 0.3 mM IPTG after the bacterial growth has attained the optical density of 0.5, 1.0 or 2.0 at 600 nm. It was observed that growth of all the three E. coli cultures were increased exponentially up to 5 h post-induction. The induction of bacterial cultures with IPTG supplementation at optical density 0.5 (OD600 ) showed maximum increase in growth as well as expression of recombinant protein in BL21CP strain followed by TB1 and DH5 strains (Fig. 2A). Recombinant BL21CPpMPI culture was induced with IPTG at OD600 0.5 and cultivated at 25 C, 30 C and 37 C for 8 h to determine the optimum growth temperature for expression of fusion protein. The results analysed by DAC-ELISA showed signicant increase in fusion protein content at 30 C followed by 25 C while minimum recovery at 37 C (Fig. 2B). The yield of specic fusion protein was signicantly higher in recombinant BL21CP strain with 24% of TSP as compared to 18% and 15% TSP obtained in TB1 and DH5 strains respectively at 30 C post IPTG induction (Fig. 2C). Recombinant BL21CP culture was grown at different pH of LB medium ranging from pH 6.0 to 9.0. The results on recovery of fusion protein showed maximum yield of 24% TSP at pH 7.0 which decreased signicantly to 1214% TSP at pH above 8.0 (data not shown). Maximum induction of fusion protein of

87.5 kDa in recombinant BL21CPpMPI culture was induced with IPTG at 30 C after 6 h incubation as revealed by SDS-PAGE analyses (Fig. 2D). The yield of fusion protein did not increase signicantly beyond 68 h of incubation. The size of fusion protein on SDS-PAGE was approximately 87.5 kDa, which matched well with sum of the theoretical mass of non-glycosylated 1 -PI (45.0 kDa) and MBP (42.48 kDa). 3.3. Purication of recombinant 1 -PI protein Recombinant 1 -PI protein and its variants were puried up to homogeneity as evident from SDS-PAGE and Western immunoblot with maximum yield of about 79 mg from 1 l of recombinant BL21CP culture, grown and induced under optimized conditions. The MBP1 -PI fusion protein was puried from cell-free extracts of bacteria by afnity chromatography on amylose resin. Majority of the fusion protein was eluted in rst few fractions after the addition of maltose in extraction buffer. SDS-PAGE of eluted protein fraction showed one major band of 87.5 kDa and another minor band of 70.0 kDa, while the immunoblot analysis conrmed the 87.5 kDa band as MBP1 -PI fusion protein (Fig. 3A, B). The minor band of 70 kDa may be an intermediate or degradation product of fusion protein. The puried fusion protein was digested with factor Xa protease that showed the appearance of only two protein bands which migrated with apparent molecular masses of 45.0 and 42.5 kDa on SDS-PAGE corresponding to 1 PI and MBP respectively (Fig. 3A). Immunoblotting of above gel with anti-1 -PI and anti-MBP antibodies further conrmed the cleavage of fusion protein into 1 -PI and MBP (Fig. 3B, C). Fusion protein of MBPParamyocin Sal of 70.2 kDa as internal control was also digested with factor Xa, which produced two proteins of molecular mass 42.5 and 27.7 kDa corresponding to MBP and Paramyocin Sal respectively that reected the efcacy and specicity of factor Xa cleavage (Fig. 3A). The factor Xa digested fusion protein was again chromatographed on second amylose column, which in turn binds with cleaved MBP fusion tag while unbound pure 1 -PI was

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Fig. 3. Characterization of MBP1 -PI fusion protein expressed in BL21CP at 30 C. (A) Coomassie brilliant blue stained 10% SDS-PAGE gel. Lane 1, prestained molecular mass standard; lane 2, amylose afnity chromatography puried fusion protein of 87.5 kDa; lane 3, puried MBP1 -PI fusion protein digested with factor Xa protease showing cleaved protein of 45.0 kDa 1 -PI and 42.5 kDa MBP respectively; lane 4, fusion protein MBPparamyocin Sal (70.2 kDa) digested with factor Xa as control showing two cleaved protein bands of 42.5 kDa MBP and 27.7 kDa paramyocin Sal respectively; lane 5, pure human 1 -PI of 52.0 kDa and MBP of 42.5 kDa. (B) Western immunoblot of the gel A with anti-human 1 -PI antibody exhibiting cross-reaction with; lane 1, fusion protein; lane 2, 1 -PI fragment of factor Xa digested fusion protein; lane 3, no cross-reaction with MBP and paramyosin proteins; lane 4, pure human 1 -PI protein. (C) Western immunoblot of the gel A with anti-MBP antibody exhibiting cross-reaction with; lane 1, 87.5 kDa fusion protein; lane 2, MBP fragment of factor Xa digested fusion protein; lane 3, MBP fragment in factor Xa digested control substrate MBPparamyocin Sal; lane 4, pure MBP protein.

Fig. 4. Purication of recombinant 1 -PI protein expressed in BL21CP strain. (A) Lane 1, prestained molecular mass standard; lane 2, IPTG induced crude cell-free extract of recombinant BL21CP harbouring pMAL-c2X vector without 1 -PI gene; lane 3, induced recombinant BL21CPpMPI extract showing expression of fusion protein of 87.5 kDa. (B) Coomassie brilliant blue stained 10% SDS-PAGE gel. Lane 1, prestained molecular mass standard; lane 2, amylose afnity puried fusion protein of 87.5 kDa; lane 3, factor Xa digested fusion protein showing 45.0 kDa 1 -PI and 42.5 kDa MBP; lane 4, puried 1 -PI protein of 45.0 kDa eluted from second amylose column; lane 5, puried human 1 -PI protein of 52.0 kDa. A minor band of 70.0 kDa was also observed (shown by arrow), which was co-puried with fusion protein and subsequently removed after second afnity chromatography. (C) Western immunoblot of the gel B with anti-human 1 -PI antibody. Lane 1, exhibiting cross-reaction with 87.5 kDa fusion protein; lane 2, 1 -PI fragment in factor Xa digested fusion protein; lane 3, puried recombinant 1 -PI protein; lane 4, pure human 1 -PI protein. Arrow corresponds to molecular mass of puried fusion, human and recombinant 1 -PI protein of 87.5, 52.0 and 45.0 kDa respectively.

collected in the ow-through (Fig. 4AC). The stepwise recovery of recombinant 1 -PI protein from 1 l of recombinant BL21CP culture is summarized in Table 1. Western immunoblot analysis of puried protein samples with anti-1 -PI antibody showed crossreaction with single discrete band of 45.0 kDa, which matched well with the theoretical molecular mass of non-glycosylated 1 Table 1 Purication steps of recombinant 1 -PI from E. coli BL21CP straina . Purication step Protein content (mg) Total soluble protein Cell-free extract First amylose afnity chromatographyf Second amylose afnity chromatographyg
a b c d e f g b

PI and reected signicant homogeneity of the puried protein (Fig. 4C). The nal yield of pure recombinant 1 -PI was about 7 mg from 1 l of bacterial culture (5.2 g wet cell weight). Similar purication prole and yields ranging between 7 and 9 mg 11 bacterial cultures were obtained for different variants of recombinant 1 -PI protein.

Specic activitye Recombinant 1 -PI 33.50 29.13 7.25

c

Yield (%)

Biologically active 1 -PI 8.80 7.21 3.77

144.0 38.30 8.38

0.06 0.19 0.45

100.00 87.00 21.6

Starting with 1 l of bacterial culture grown at 30 C for 6 h after IPTG induction. Total soluble protein determined by Bradford assay. Recombinant protein estimated by DAC-ELISA using anti 1 -PI antibody. Biological activity (elastase inhibitory capacity) estimated by residual PPE activity assay. Specic activity is based on amount of biologically active 1 -PI per mg of total soluble protein. Puried MBP1 -PI fusion protein. Factor Xa cleaved puried recombinant 1 -PI protein.

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Fig. 5. Identication and characterization of 1 -PI protein by MALDITOF/TOF. (A) The observed MS spectrum (peptide mass ngerprint) of the tryptic digest of recombinant 1 -PI puried from E. coli. Further MS/MS analysis and peptide sequencing was performed by selecting at least ten precursor ions (data not shown). Inset shows the observed peak of 44328.35 Da for mass of singly charged ion species (M+H)+ of intact protein, which corresponds to theoretical mass of unglycosylated 1 -PI. (B) The observed MS spectrum of the tryptic digest of pure human serum 1 -PI protein. Inset shows the observed molecular mass of 49824.49 Da for intact protein, which is 2 kDa less than theoretical mass (52 kDa) of glycosylated human 1 -PI. The decrease in observed mass may be due to fragmentation of glycan chains and desialylation of the glycoprotein caused by matrix properties.

3.4. Recombinant 1 -PI protein analysis by MALDITOF/TOF Accurate masses of recombinant 1 -PI expressed in E. coli and native human serum 1 -PI protein were determined by MS in linear high-mass positive ion mode with low mass gate (LMG) set at 100 Da. The observed peaks showed molecular weights of 44328.36 Da and 49824.49 Da for puried recombinant E. coli expressed and native human 1 -PI, respectively (Fig. 5A, B-inset). The observed mass for recombinant protein was matched well with theoretical mass of unglycosylated 1 -PI, while a 2 kDa decrease in observed mass of human 1 -PI (theoretical mass 52.0 kDa) may be due to fragmentation of glycan chains and removal of sialic acid residues from the glycosylated human 1 -PI caused by hot properties of CHCA matrix. For unambiguous identication of protein, peptide fragments generated by in-gel trypsin digestion were analyzed by MS (peptide mass ngerprinting) and MS/MS (peptide sequencing) in reector positive ion mode. The resulting spectra were searched using Mascot search engine and both human and recombinant proteins were signicantly identied as 1 -PI with a MOWSE score of 416 and 590, respectively at p < 0.05 with high sequence coverage. The MS spectra of tryptic peptides of both the proteins are

shown in Fig. 5A and B. These results suggested that recombinant 1 -PI expressed in E. coli is fully intact with complete N-terminal sequence and molecular mass is similar to the native unglycosylated human protein puried from serum. 3.5. Biological activity and thermostability of recombinant 1 -PI protein Biological activity of E. coli expressed recombinant 1 -PI protein(s) was monitored by residual PPE activity assay that showed efcient inhibition of elastase activity with specic activity of about 0.45 0.04 for wild type 1 -PI after two cycles of amylose afnity chromatography. Biological activity of factor Xa cleaved recombinant 1 -PI protein was found relatively enhanced in comparison to fusion complex (Table 1). The specic activities of the cleaved 1 -PI variant proteins were signicantly enhanced up to 0.68 0.07 for 1 -PIFC, 0.61 0.05 for 1 -PIFL, 0.52 0.06 for 1 -PIAG, 0.45 0.03 for 1 -PIMV and 0.47 0.04 for 1 -PIMI, which suggest the stabilizing effects of incorporated mutations in the expressed protein (Table 2). Thermal stability analysis of the puried wild type recombinant 1 -PI and variant 1 -PI proteins was performed at 37 C and

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Table 2 Yield, recovery and specic activities of puried recombinant 1 -PI and variants from E. colia . Construct 1 -PI variants Protein content (mg) Total soluble protein pMPI pMFC pMFL pMAG pMMV pMMI
a b c d e b

Specic activitye Recombinant 1 -PI 7.25 8.06 8.74 7.41 6.75 8.12 0.28 0.08 0.11 0.17 0.23 0.22
c

Biologically active 1 -PI 3.77 6.45 6.27 4.53 3.57 5.01 0.34 0.66 0.51 0.52 0.24 0.43

WT F51C F51L A70G M358V M374I

8.38 9.48 10.28 8.71 7.94 10.64

0.45 0.68 0.61 0.52 0.45 0.47

0.04 0.07 0.05 0.06 0.03 0.04

Determined for factor Xa cleaved recombinant 1 -PI protein(s) after nal purication step. Total soluble protein determined by Bradford assay. Recombinant protein estimated by DAC-ELISA using anti 1 -PI antibody. Biological activity (elastase inhibitory capacity) estimated by residual PPE activity assay. Specic activity is based on amount of biologically active 1 -PI per mg of total soluble protein.

54 C. Most mutant proteins except 1 -PIMV showed signicantly enhanced stability at 37 C over time as compared to the wild type recombinant 1 -PI. Only 20% loss in activity was observed for mutant variants after 150 min of incubation as compared to >35% and 80% loss of protein activity in M358 V variant and 1 -PIWT respectively, while activity of puried glycosylated human native 1 -PI remained unaffected (Fig. 6A). The results of heat inactivation of protein activity at 54 C showed that activity of recombinant wild type 1 -PI was 50% inhibited after 7.5 min of incubation and completely lost after 30 min of treatment, whereas recombinant 1 -PI variant proteins showed signicantly enhanced resistance to thermal inactivation in comparison to wild type 1 -PI (Fig. 6B). The variant 1 -PI protein having F51C substitution showed maximum protection to thermal inactivation exhibiting 50% inhibition of activity after 24 min of incubation, followed by 1 -PI substituted with F51L, A70G, M374I and M358 V, respectively. Retention of maximum biological activity after 60 min of incubation at 54 C was obtained in variant protein 1 -PIFC (11%), followed by 1 -PIFL (10.2%), 1 -PIAG (9.8%), 1 -PIMI (7.1%) and 1 -PIMV (1.4%), while

glycosylated native human serum 1 -PI lost only 20% of its activity after 60 min of treatment (Fig. 6B). 4. Discussion We have demonstrated high-level expression of plant codonoptimized modied synthetic 1 -PI gene and its variants consisting rare codons in E. coli under optimized culture conditions. The modied 1 -PI genes were expressed as MBP fusion protein, driven by ptac promoter for high-level expression together with overcoming the limitations of insolubility, misfolding-mediated instability, degradation or aggregation of recombinant foreign protein (Kapust and Waugh, 1999). Comparative expression prole showed consistently maximum expression of recombinant 1 -PI and its variant proteins (2224% of TSP) in BL21CP strain of E. coli. This strain carries extra copies of tRNA genes for arginine, isoleucine and leucine, therefore, may augment for rare codons in the modied 1 -PI gene and leads to higher level of expression. Similar pattern of expression was observed when a plant codon-optimized synthetic cry gene of Bacillus thuringiensis was expressed as NusA fusion protein in E. coli carrying copies for rare tRNA codons (Kumar et al., 2005). In addition, we have also observed expression of fusion protein up to 18% and 15% of TSP in TB1 and DH5 strains respectively. This level of recombinant 1 -PI expression in E. coli strains without any extra copies of tRNA genes could be attributed to the optimum culture conditions and also to the presence of N-terminal MBP fusion tag that increases stability of expressed mRNA, translational efciencies (Kapust and Waugh, 1999; Lian et al., 2009) and also protecting the passenger protein from intracellular proteolysis (Bach et al., 2001). Similar kind of observations has been reported owing to N-terminus addition of GST fusion tag that signicantly increased the expression of genes containing 33% of rare codons (Wu and Oppermann, 2003; Wu et al., 2004). The N-terminal fusion of MBP with 1 -PI coding sequence leads to late emergence of rare codon that mimics stabilizing effect for translational complex formation and allows formation of specic secondary structure for increased gene expression (Goldman et al., 1995; Gursky and Beabealashvilli, 1994). Over expression of ScFv fraction of antibodies and other heterologous proteins in E. coli as MBP or GST fusion, however, support our results and hypothesis (Bird et al., 2004; Esposito and Chatterjee, 2006; Bach et al., 2001). Determination of exact molecular mass and primary structure of the expressed recombinant protein is essential for molecular identication and characterization. Our results of mass spectrometric analyses and peptide mass ngerprinting of the puried recombinant 1 -PI expressed in E. coli and native human serum 1 -PI showed fully intact state of recombinant 1 -PI protein with identical N-terminal sequence and molecular mass. These results reect the structural integrity of the recombinant 1 -PI expressed in E. coli and retention of corresponding specic biological activity, which

Fig. 6. Thermostability analysis of E. coli expressed recombinant 1 -PI protein and its variants at 37 C and 54 C. (A) Inactivation curves of native human serum 1 -PI and cleaved 1 -PI protein(s) puried from recombinant BL21CP having wild type expression vector (pMPI) and vectors having single point mutations (pMFC, pMFL, pMAG, pMMV and pMMI) at 37 C. (B) Heat inactivation analysis at 54 C for the same. Approximately 5 g of puried 1 -PI protein was used in each sample.

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are the critical parameter for therapeutic application of recombinant proteins (Karnaukhova et al., 2006; Hepner et al., 2005). Major limitations for expression of heterologous proteins in E. coli are misfolding-mediated aggregation, formation of insoluble inclusion bodies, loss of specic biological activity and instability due to lack of post-translational modications (Makrides, 1996; Bird et al., 2004; Lian et al., 2009). Expression of human gene for 1 PI in E. coli has been attempted earlier to achieve biologically active protein. However, activity of recombinant 1 -PI protein expressed in E. coli was decreased due to protein oligomerization (Kwon et al., 1995), N-terminal extension of the protein (Courtney et al., 1984), or very complex purication procedure (Bischoff et al., 1991). We have expressed recombinant 1 -PI in E. coli strain BL21CP as MBP fusion protein and achieved higher expression (upto 24% of TSP), with enhanced solubility, retention of structural integrity, high biological activity and rapid purication on amylose afnity column. The puried recombinant 1 -PI was unglycosylated and less stable but showed high biological activity since glycosylation is not required for its activity (Courtney et al., 1984). We have developed ve variants of the protein with single amino acid substitutions at critical domains to increase the stability of the molecule. The in-vivo half-life of unglycosylated protein can also be increased by PEGylation that prevents rapid clearance from the blood after intravenous infusion and decreases immunogenicity of therapeutic proteins (Cantin et al., 2002). Enhanced stability, structural integrity and functional efcacy of recombinant 1 -PI and other therapeutic proteins expressed in alternate host are of major concern and always in high demand to decipher the mechanism of proteinase inhibition, folding, misfolding and aggregation for developing therapeutic strategies (Cabrita and Bottomley, 2004). The native form of serine protease inhibitors (serpins) exist in metastable strained state that reect on the efcacy of their biological activity. However, this could be relieved by incorporation of different single amino acid substitutions at specic sites for more stable conformation of serpins (Im et al., 2004). Therefore ve variants of 1 -PI protein were developed via specic point mutations in strategic domains to relieve the strained state of native 1 -PI protein. Phe51 and Met374 residues lie in the breach domain as strands of sheet-B at hydrophobic core of the protein molecule. Substitution of these positions with smaller linear aliphatic residues like Cys, Leu and Ileu would decrease the size of chains around hydrophobic core of 1 -PI and allow more freedom and improved tertiary packing preventing opening of sheet-A and RCL insertion (Kwon et al., 1994; Im et al., 2004). This would eventually increase the stability of native 1 -PI protein. Increased backbone freedom is another option for stabilization state of inhibitory serpins. Ala70 is located at the beginning of Chelix and its substitution with small exible residue like Gly may result in better packing of proximal residues, thereby increasing thermostability of serpins by releasing the energy constraint associated with Ala70 (Im et al., 2004). Oxidation of Met358 residue located at P1 position of RCL domain of 1 -PI results into signicant loss of inhibitory activity for elastase, therefore, replacement of Met358 with Val, which is relatively refractory to oxidation should improve efciency for elastase inhibition (Travis et al., 1985; Levina et al., 2009). Most of the substitutions engineered in serpins to improve their stability and efciency for proteolytic destruction of elastase were resulted into increased thermostability as well. Considering this as an important parameter we have compared thermostability of wild type and variant 1 -PI molecules and our results are in agreement with earlier reports and suggest signicance of point mutations at critical sites to improve the biological activity and stability of 1 -PI (Kwon et al., 1994; Im et al., 2004). To conclude, our results have demonstrated high-level expression and simple purication of stable and biologically active recombinant 1 -PI protein and its variants from E. coli. This

approach is quick, cost-effective and especially suitable for designing expression strategies in structural and functional genomics for expressing therapeutic proteins in alternative heterologous hosts. In addition, with the availability of extensive studies on different mutated 1 -PI, it will be possible to understand the molecular mechanisms of proteinase inhibition, folding, misfolding and aggregation towards developing therapeutic strategies. This type of protein engineering opens a new dimension in the area of therapeutic protein production with increased stability and efcacy. Acknowledgements We are grateful to Director, NBRI, Lucknow for infrastructural support, encouragement and valuable suggestions. We thankfully acknowledge Council of Scientic and Industrial Research (CSIR), India for providing funds and senior research fellowships to SA and SJ. This work was carried out under the CSIR Network Project CMM 0004. References
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