1 Western Regional Research Center, USDA-ARS, Albany, California94710.

2 To whom correspondence should be addressed.

2330277-8033/01/0300-0233$19.50/0 © 2001 Plenum Publishing Corporation

An E. coli Expression System for the Extracellular Secretionof Barley a-Amylase

Charles C. Lee,1 Dominic W. S. Wong,1,2 and George H. Robertson1

Received April 15, 2001

Libraries of modified genes are often screened during the process of genetically engineering en-zymes with specifically tailored activities. It is important, therefore, to create expression systemswhich allow for the rapid screening of many clones. We developed an Escherichia coliexpressionsystem which will secrete enzymes into the growth medium. We describe the first reported expres-sion of barley a-amylase in E. coli. The enzyme is secreted onto solid media containing starch toproduce easily visualized halos. In addition, the enzyme is secreted into liquid media in an intact,active form.

KEY WORDS: Secretion; a-amylase; barley; E. coli.

Journal of Protein Chemistry, Vol. 20, No. 3, 2001

both yeast and B. subtilisthat can direct the extracellularsecretion of a recombinant enzyme (Castelli et al.,1994;Cregg et al.,1993; Hemila et al.,1992; Lam et al.,1998;Scorer et al., 1993). These signal sequences are excisedfrom the recombinant protein during secretion. In con-trast, E. coli does not have such a well-defined excisablesignal sequence which will direct secretion of the proteinto the growth medium. However, E. coli does have ad-vantages over yeast and B. subtilis.It has a much highertransformation efficiency than B. subtilisand yeast, andthus a larger population of the library of gene mutantscan be screened. In addition, E. coligrows at a faster ratethan yeast, thus ensuring a high-throughput screen. Fi-nally, plasmid preparations from E. coli are simpler andresult in much higher DNA yields, allowing for the di-rect characterization of clones. In contrast, yeast plas-mids need to be amplified in bacteria.

We have developed an E. coli expression systemthat secretes recombinant enzyme into the growthmedium. We present the first report of the successfulE. coli expression and secretion of active barley low-pI a-amylase. This enzyme is critical to the brewing

1. INTRODUCTION

One of the main strategies for improving the efficienciesof biochemical industrial processes is the development ofrecombinant enzymes that possess novel or enhanced ac-tivities. A common method for engineering better en-zymes is by random mutagenesis of the correspondinggene followed by screening of expression libraries to iso-late the desired mutants. Many methods exist to generatemutations in the gene of interest. Error-prone PCR3 is fre-quently used to create libraries of mutants (Cadwell andJoyce, 1992). In addition, the advent of several “DNAshuffling” strategies has dramatically improved the gener-ation of modified enzymes by recombining multiple use-ful mutations (Judoet al.,1998; Shaoet al.,1998; Stem-mer, 1994; Zhaoet al., 1998). Once a library of mutantgenes has been obtained, it can be screened, and the cloneswith the desired activities can be isolated and subjected toadditional rounds of mutation and selection.

A limiting step is the expression screen. In general,the library is transformed into a microorganism, such asyeast, Bacillus subtilis,or Escherichia coli.There areseveral signal sequences that have been characterized in

3 Abbreviations: BRP, Bacteriocin-release protein; HlyA, hemolysin;IPTG, isopropyl-b-D-thiogalactopyranoside; kil, colicin-release pro-tein; MOPS, 3-(N-morpholio)butanesulfonic acid; PCR, polymerasechain reaction; pelB, pectate lyase B.

234 Lee, Wong, and Robertson

industry and has been the focus of many studies. Whencolonies were grown on solid media containing starch,the amylase was secreted, and the enzyme digested thestarch to form easily visualized halos. In liquid culture,the barley amylase was secreted into the media in afull-length active form.

2. MATERIALS AND METHODS

2.1. Plasmid Construction

The pSE420-gIII expression vector was generatedfrom portions of the pSE420 and pBAD-gIIIB plasmids(Invitrogen). The gene III signal sequence and the poly-linker sequence were PCR-amplified from pBAD-gIIIBusing PfuTurbo DNA polymerase (Stratagene) and theprimers 58-AAAAAACTGCTGTTCGCGATTCCG-38and 58-GTATGCGGCCGCGGCAAATTCTGTTTTAT-CAGACCGCTTC-38. The resulting PCR product wasdigested withNotI to produce a DNA fragment with oneblunt and one staggered end. This fragment was clonedinto the pSE420 vector backbone which had been treatedwith NcoI, then T4 DNA polymerase (to create bluntends), and finallyNotI (to generate a staggered end).

The pSE420-gIII–LpI amylase expression vectorwas generated by PCR amplification of the barley low-pIa-amylase gene [isolated from a barley cDNA library(Strategene)] with PfuTurbo DNA polymerase and theprimers 58-GGCGGTACCCATATGCACCAAGTCCT-CTTTCAGGG-38 and 58-GGCTCTAGACTGCAGT-CAGCTCCGTTGTAGTGTTGC-38. The PCR productwas then digested with KpnI and XbaI and cloned intothe pSE420-gIII backbone that was also digested withKpnI and XbaI.

All enzymes were purchased from New EnglandBiolabs unless otherwise stated.

2.2. Expression of Barley a-Amylase

To express the barley amylase enzyme, the pSE420-gIII–LpI plasmid was transformed into the E. coli strainBL21-Gold (Stratagene). To observe amylase secretiononto solid media, individual clones were spotted onto LBagar plates (Sambrook et al., 1989) that were supple-mented with 0.5% starch (Sigma), 50 mM MOPS (pH7.0), and 0.1 mM isopropyl-b-D-thiogalactopyranoside(IPTG). Because the barley low-pI amylase requirescalcium for stability and activity (Bush et al., 1989),3.5 mM CaCl2 was also added to the media. The plateswere incubated at 30°C overnight and then stained withiodine vapor to visualize the regions of starch digestion.

To analyze amylase secretion into liquid media, in-dividual colonies were inoculated and grown at 25°C inLB broth (Sambrook et al.,1989) that was supplementedwith 50 mM MOPS (pH 7.0), 1 mM IPTG, and 10 mMCaCl2. A titration of various concentrations of CaCl2

(3.5–25mM) had demonstrated that a higher amount ofCaCl2 was required in the liquid media as compared tothe solid for the maximum level of activity.

At various time intervals, aliquots of the bacterialliquid culture were removed and spun down, and themedia and whole-cell fractions were collected. The rela-tive levels of amylase enzyme were determined by West-ern blot analysis. Equivalent culture volumes of mediaand whole-cell fractions were electrophoresed through a10% SDS–PAGE gel. The protein was then transferredto nitrocellulose membrane (Schleicher & Schuell), andthe blot was sequentially probed with anti-amylase anti-body and anti-rabbit HRP antibody (Biorad). The proteinbands were visualized using the Lumi-Light WesternBlotting Substrate (Boehringer Mannheim) according tomanufacturer’s instructions. The intensities of the pro-tein bands were quantified on a gel imaging system(Alpha Innotech, San Leandro, CA).

2.3. Measurement of Amylase Activity

Amylase activity in the liquid media was measuredusing slight modifications of a previously published pro-tocol (Wong et al.,2000). In brief, 160 ml of culture su-pernatant was added to 56 ml of a solution of Phadebas,a dye-labeled starch substrate (Pharmacia & Upjohn),and incubated at 37°C for 30 min. The reaction was thenspun down, and 150 ml of the supernatant was measuredat 620 nm on a 96-well microplate scanner (Labsystems,Vista, CA). Enzyme activity units were determined byplotting a standard curve using purified endogenous bar-ley a-amylase of known activity.

3. RESULTS

In order to express and secrete the barley low-pIa-amylase enzyme extracellularly fromE. coli, an ex-pression vector (pSE420-gIII) was created which con-sisted of the pSE420 vector backbone with the gene IIIsignal sequence tag and polylinker from the pBAD-gIIIB plasmid (see Section 2). Gene III encodes a cap-sid protein of theE. coli filamentous phage fd, whichis assembled at the bacterial membrane (Schalleret al., 1978). When the gene III protein is secreted

from the cytoplasm, the signal peptide comprising theN-terminal 18 amino acids is cleaved from the protein(Goldsmith and Konigsberg, 1977). When the gene IIIsignal sequence is fused to a recombinant enzyme, itcan direct secretion of the protein out of the cytoplasmand then be cleaved. Thus, any activity interferencecaused by extraneous amino acid residues should beminimized.

Transcription of the fusion protein from pSE420-gIII is controlled by the lac operator and trc promoter,which is inducible by IPTG, a nonhydrolyzable lactoseanalog. Transcription from the original pBAD-gIIIBplasmid is driven by the araBAD promoter, which wasactivated by arabinose. In order to achieve a steady levelof gene expression from pBAD-gIIIB, a constant intra-cellular level of arabinose must be maintained. Thus, itis important to use a bacterial strain which is unable tometabolize arabinose, such as TOP10. This requirementdramatically limits the number of bacterial strains thatcan be used with the pBAD-gIIIB vector. With the in-creased flexibility provided by the IPTG-inducibility ofpSE420-gIII, 18 different E. colistrains were screened todetermine which would give the least amount of back-ground activity (i.e., bacterial carbohydrase activity) onour starch plate halo assay (Table I). The BL21-Gold cellline was chosen because this strain gave little back-ground in the starch activity assay and had very hightransformation efficiency.

The barley low-pIa-amylase gene was subclonedinto the pSE420-gIII vector (see Section 2). BL21-Goldcells were transformed with either the pSE420-gIII vec-

tor or the pSE420-gIII–LpI amylase expression con-struct. To determine whether the amylase enzyme couldbe secreted onto solid media, individual colonies werespotted on an LB agar plate containing 0.5% starch and0.1 mM IPTG. After an overnight incubation at 30°C,the colonies containing the pSE420-gIII–LpI plasmidclearly secreted amylase enzyme as evidenced by thehalo of digested starch around the colonies (Fig. 1). In-cubation at 37°C resulted in smaller halos (data notshown).

The formation of the halos resulting from starch di-gestion indicated that the expression system could beused to screen libraries. However, it was also importantto demonstrate that the amylase enzyme was secretedinto the media in liquid cultures. Amylase that is se-creted into the media can be readily purified so thatdifferences in the specific activities among promisingmutant enzymes can be determined. BL21-Gold cellstransformed with either the pSE420-gIII vector or thepSE420-gIII–LpI expression plasmid were grown in liq-uid culture containing 1 mM IPTG at 25°C. It was nec-essary to buffer the growth media with 50 mM MOPS(pH 7.0) to observe amylase activity. Without buffering,the pH of the culture media exceeded 8.5, which was in-compatible with the amylase activity (Sogaard andSvensson, 1990).

At different time points, aliquots of the culturestransformed with either the pSE420-gIII or the pSE420-gIII–LpI plasmid were spun down, and the media super-natant was assayed for activity (Fig. 2). Media from theculture containing cells transformed with the amylase ex-pression vector showed increasing levels of enzyme ac-tivity up to 48 h. The vector control culture showed noactivity.

To determine the relative amount of amylase thatwas secreted into the liquid media, Western blot analysiswas conducted on samples of the culture supernatant and

E. coli Expression of Barley a-Amylase 235

Table I. Analysis of Background Carbohydrase Activity fromVarious E. coli Cell Linesa

Cell line Activity Cell line Activity

AD494(DE3)4 11 JM1011 111

BL21-Gold5 2 JM1091 11

BL215 2 LMG1943 111

BL21(DE3)5 2 MC10611 11

BL21(DE3)pLysE4 2 RR11 2

BL21(DE3)pLysS5 2 Top103 11

DH5a1 1 TST3-MC41006 2

FB22 111 XLmutS5 11

HB1011 2 XL1 Blue (MRF8)1 11

a Individual bacterial colonies were spotted and grown overnight on LBagar plates containing 1% starch and 1 mM IPTG. Plates were stainedwith iodine vapor to detect background starch degradation. “2” indi-cates very little starch degradation. “1” indicates increasing levels ofstarch degradation. Strains were obtained from 1ATCC, 2E. coli Ge-netic Stock Center (New Haven, CT), 3Invitrogen, 4Novagen, 5Strata-gene, and 6Dr. T. Silhavy (Princeton University).

Fig. 1. Starch digestion on solid media. E. coli clones transformedwith either pSE420-gIII vector (top row) or pSE420-gIII–LpI amylaseexpression vector (bottom row) were spotted onto LB agar platescontaining starch. After overnight incubation at 30°C, starch digestionwas visualized by staining plates with iodine vapors.

4. DISCUSSION

In summary, the pSE420-gIII expression vectorprovides a vehicle to rapidly screen enzyme variants. Wedemonstrate that active barley low-pI a-amylase enzymecan be secreted extracellularly from E. coli. Previous re-ported attempts to express the barley low-pI a-amylasein E. coli were unsuccessful (Sogaard, 1989). Althoughthis enzyme has also been expressed and secreted fromyeast (Juge et al., 1993, 1996; Sogaard et al., 1991; So-gaard and Svensson, 1990) and Aspergillus niger(Jugeet al., 1998), using E. coli offers great advantages interms of higher transformation efficiencies, faster growthrates, and direct clone analysis without the need for anintermediate host to amplify the plasmid.

We used the gene III signal sequence to secrete re-combinant protein from the cell. There are many othermeans to release E. coli-produced proteins that aretrapped in the cell, but these methods all have draw-backs. One approach is to use external physical force.For example, exposing the cells to a high-voltage elec-trical (Ohshima et al., 2000) or osmotic (Ausubel et al.,1999) shock has proven successful in releasing recombi-nant proteins from E. coli. Unfortunately, these tech-niques are unwieldy to use on a large-scale expressionscreen and will not work on an LB agar assay. The abil-ity of the gene III signal sequence to direct extracellularsecretion on solid media dramatically increases thethroughput of an expression screen.

Another strategy that has been employed is the useof proteins that permeabilize the E. coli cell membrane,such as bacteriocin-release protein (BRP) (van der Walet al., 1995) and colicin-release protein (kil) (Robbenset al., 1995). We tested whether BRP would release thebarley low-pI a-amylase. An expression vector carryingthe a-amylase gene was cotransformed with the BRP ex-pression vector pSW1 (Display Systems Biotech, Vista,CA). Although the BRP did release the a-amylase en-zyme, there was significant background from the vectorcontrols (data not shown). Presumably, BRP was nonse-lectively releasing many E. colicellular proteins that hadcarbohydrase activity. In addition, cell growth was im-paired. Attempts at titrating the amount of BRP produceddid not resolve these problems.

Another technique for secreting protein into thegrowth medium involves the use of the E. colihemolysin(HlyA) secretion system (Hahn et al., 1998; Jarchauet al.,1994; Kern and Ceglowski, 1995). The HlyA sig-nal sequence directs a fusion protein through both theinner and outer membranes in a one-step process, thuseliminating the common problem of protein entrapmentin the periplasm. One drawback to this method is that theHlyA signal sequence is not excised from the fusion pro-

236 Lee, Wong, and Robertson

Fig. 2. Amylase activity in liquid media. Cultures ofE. coli trans-formed with either pSE420-gIII vector (open circles) or pSE420-gIII–Lpl amylase expression vector (closed circles) were grown at 25°C. Atvarious time intervals, aliquots of the culture media were collected andassayed for activity (international enzyme units).

Fig. 3. Western blot of E. coli culture fractions. Aliquots (100 ml) ofE. coli culture transformed with either pSE420-gIII vector (Vec) orpSE420-gIII–LpI amylase expression vector (Amy) were harvested at24 hr, and the samples were spun down. The medium supernatant wasseparated from the cell pellet, which was resuspended in 100 ml of SDSsample buffer. Two ml of the medium (M) and the resuspended cellpellet (C) were subjected to SDS–PAGE on a 10% gel, blotted ontonitrocellulose membrane, and probed with anti-amylase antibody.

the whole-cell fraction (Fig. 3). Approximately 65% ofthe amylase enzyme was secreted into the liquid media.In addition, the enzyme was primarily full-length, andvery little proteolysis was evident.

tein, so there could be interference of the enzyme activ-ity by these residues. In this regard, our use of the geneIII signal sequence which is excised by the cellular ma-chinery is a distinct advantage.

Others have used excisable signal sequences inE. coli to secrete protein. For instance, proteins fused withthe pelB signal sequence were secreted into the growthmedium at relatively high levels (Kipriyanov et al.,1997; Lucic et al., 1998). However, when we tested theability of the pelB signal sequence to secrete recombi-nant barley low-pI a-amylase, we were unable to detectany activity (data not shown). Thus, the pSE420-gIIIvector, which employs the gene III signal sequence,serves as a useful alternative general strategy, especiallywhen other E. coli excisable tags fail to direct extracel-lular recombinant protein secretion.

ACKNOWLEDGMENTS

We thank members of the D. W. S. Wong labora-tory for critical reading of the manuscript and discus-sions. We also thank R. L. Jones and P. C. Bethke for thekind gift of a-amylase antibody and T. Silhavy for pro-viding the TST3-MC4100 cell line.

REFERENCES

Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman,J. G., Smith, J. A., and Struhl, K. (eds.). (1999). Short Protocolsin Molecular Biology,J Wiley, New York.

Bush, D. S., Sticher, L., van Huystee, R., Wagner, D., and Jones, R. L.(1989). J. Biol. Chem.264, 19392–19398.

Cadwell, R. C. and Joyce, G. F. (1992). PCR Meth. Appl.2, 28–33.Castelli, L. A., Mardon, C. J., Strike, P. M., Azad, A. A., and

Macreadie, I. G. (1994). Gene142, 113–117.Cregg, J. M., Vedvick, T. S., and Raschke, W. C. (1993). Biotechnol-

ogy (NY) 11, 905–910.

Goldsmith, M. E. and Konigsberg, W. H. (1977). Biochemistry16,2686–2694.

Hahn, H. P., Hess, C., Gabelsberger, J., Domdey, H., and von Specht,B. U. (1998). FEMS Immunol. Med. Microbiol.20, 111–119.

Hemila, H., Pakkanen, R., Heikinheimo, R., Palva, E. T., and Palva, I.(1992). Gene116, 27–33.

Jarchau, T., Chakraborty, T., Garcia, F., and Goebel, W. (1994). Mol.Gen. Genet.245, 53–60.

Judo, M. S., Wedel, A. B., and Wilson, C. (1998). Nucleic Acids Res.26, 1819–1825.

Juge, N., Sogaard, M., Chaix, J. C., Martin-Eauclaire, M. F., Svensson,B., Marchis-Maouren, G., and Guo, X. J. (1993). Gene 130,159–166.

Juge, N., Andersen, J. S., Tull, D., Roepstorff, P., and Svensson, B.(1996). Protein Expr. Purif.8, 204–214.

Juge, N., Svensson, B., and Williamson, G. (1998). Appl. Microbiol.Biotechnol.49, 385–392.

Kern, I. and Ceglowski, P. (1995). Gene163, 53–57.Kipriyanov, S. M., Moldenhauer, G., and Little, M. (1997). J. Immunol.

Meth.200, 69–77.Lam, K. H., Chow, K. C., and Wong, W. K. (1998). J. Biotechnol.63,

167–177.Lucic, M. R., Forbes, B. E., Grosvenor, S. E., Carr, J. M., Wallace,

J. C., and Forsberg, G. (1998). J. Biotechnol.61, 95–108.Ohshima, T., Hama, Y., and Sato, M. (2000). Biochem. Eng. J.5,

149–155.Robbens, J., Raeymaekers, A., Steidler, L., Fiers, W., and Remaut, E.

(1995). Protein Expr. Purif.6, 481–486.Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular

Cloning,Cold Spring Harbor Laboratory Press, Cold Spring Har-bor, New York.

Schaller, H., Beck, E., and Takanami, M. (1978). In The Single-Stranded DNA Phages(Denhardt, D. T., Dressler, D., and Ray,D. S., eds.), Cold Spring Harbor Laboratory Press, Cold SpringHarbor, New York.

Scorer, C. A., Buckholz, R. G., Clare, J. J., and Romanos, M. A.(1993). Gene136, 111–119.

Shao, Z., Zhao, H., Giver, L., and Arnold, F. H. (1998). Nucleic AcidsRes.26, 681–683.

Sogaard, M. (1989). M. Sc. Thesis, University of Copenhagen.Sogaard, M. and Svensson, B. (1990). Gene94, 173–179.Sogaard, M., Olsen, F. L., and Svensson, B. (1991). Proc. Natl. Acad.

Sci. USA88, 8140–8144.Stemmer, W. P. (1994). Nature370, 389–391.van der Wal, F. J., Luirink, J., and Oudega, B. (1995). FEMS Micro-

biol. Rev.17, 381–399.Wong, D. W. S., Batt, S. B., and Robertson, G. H. (2000). J. Agric.

Food Chem.48, 4540–4543.Zhao, H., Giver, L., Shao, Z., Affholter, J. A., and Arnold, F. H.

(1998). Nat. Biotechnol.16, 258–261.

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