Hyper expression of an environmentally friendly synthetic polymer gene

BIOTECHNOLOGY LETTERS Volume I7 No.7 (July 1995) pp.745-750 Received 17th May

I-IYPER EXPRESSION OF AN ENVIRONMENTALLY POLYMER GENE

FRIENDLY SYNTHETIC

C. Guda’, X. Zhang’, D.T. McPherson’, J. Xu’, J.H. Cherry’, D. W. Urr$ and H. Daniell” 1Molecular Genetics Program, Department of Botany and Microbiology, 101, Lie Sciences Building, Auburn University, Auburn, AL 36849-5407 *Laboratory of Molecular Biophysics, School of Medicine, The University of Alabama at Birmingham, Biigham, Alabama 35294-0019 SUMMARY

Biodegradable polymers offer an environmentally friendly alternative to petroleum-based polymers. Applications of protein based polymers include the use of these compounds in the fields of medicine, molecular-based energy conversions, the manufacture of unique fibers, coatings and biodegradable plastics. We report here expression of a synthetic gene G(VPGVG),,,-VPGV coding for the EG-120mer (elastomer) in E. coli. Polymer expression is observed in uninduced cells grown in terrific broth in polyacrylamide gels negatively stained with CuCl,. Electron micrographs reveal formation of inclusion bodies in uninduced cells occupying upto 80-90% of the cell volume under optimal growth conditions. To the best of our knowledge this report representsthe first demonstration of hyper expression of a synthetic gene (with no natural analog) in E. coli. INTRODUCTION Protein-based polymers, high molecular weight chain molecules comprised of repeating peptide sequences,provide many advantagesover the traditional petroleum-based polymers. Proteinbased polymers are environmentally clean over their entire life cycle from production to disposal; they can be produced from renewable resources using water-based processing and are biodegradable, whereas petroleum-basedpolymers derive from an exhaustible resource, require toxic and hazardous chemicals in their production and are center stage in the problem of solid waste disposal. Both classesof polymers can exist in different commercially important physical states of hydrogels, elastomers and plastics, but protein-based polymers are unrivaled in their capacity to control primary structure with about twenty different monomer units available and with a near infinite diversity of combinations of monomers. They are capable of all of the

745

functional roles of which proteins are capable in living organisms and yet they can be designed to perform functions that evolution has never called upon proteins to perform (Krejchi et al., 1994; Urry et al., 1994). Protein-based polymers have been shown to have remarkable biocompatibility (Urry et al., 1991) thereby enabling a whole range of medical applications including the prevention of post-surgical adhesions,tissuereconstruction and programmed drug delivery (Urry et al., 1993). For example, the polymer poly(GVGVP), used in this study, has been shown to prevent adhesions in the rat contaminated peritoneal model following abdominal injury (Urry et al., 1993). On the non-medical side, there are transducers, molecular machines, superabsorbents, biodegradable plastics, and controlled releaseof agricultural crop enhancementagents, such as herbicides, pesticides, growth factors and fertilizers. What is required for the commercial viability of protein-based polymers is a cost of production that would begin to rival that of petroleum-basedpolymers. The potential to do so residesin low cost bioproduction. In this report, without any need for extraneoussequencesfor purification or adequate expression (McPherson et al., 1992), we demonstrate in E. coli a dramatic hyper-expressionof an elastin protein-basedpolymer, (Gly-Val-Gly-W-Pro),

or poly

(GVGVP), which is a parent polymer for a diverse set of polymers that exhibit inverse temperaturetransitions of hydrophobic folding and assemblyas the temperatureis raised through a transition range (Urry, 1995) and which can exist in hydrogel, elastic, and plastic states. EXPERIMENTAL METHODS Vector construction and gene expression Using synthetic oligonucleotides and PCR, the gene (GVGVP),,, (McPherson et al., 1992) was amplified and inserted into pUCl18 as a BamHI fragment; (GVGVP),, PflMl gene fragment was ligated with suitable adapter oligos at different ratios determined to give concatamersof desired length and these concatamerswere subsequently cloned into pUCl18. Details of a series of these recombinant synthetic gene constructs will be published elsewhere (McPherson er al., 1995, in preparation). The protein basedpolymer gene (GVGVP),*, coding for the three amino acids glycine, valine and proline was inserted into the expressionvector pET1 Id (Novagen) as a NcoI and BamHI fragment under the control of the l7 promoter. The pETlld construct containing (GVGVP),,, coding sequencewas transformed into E. coli strain HMS174 (DE3) which carries a gene for the T7 polymerase in its genomic DNA (Studier and Moffat, 1986). Expression of an inserted foreign gene in pETlld is regulated by two LucZrepressor genes located in the plasmid pET1 Id as well as in the genomeof the host strain HMS174 (DE3). Gene expression was studied in sampleseach grown in either Luria broth (LB) or in Terrific broth (Tartof and Hobbs, 1987) in the presenceor absenceof ampicillin (100 pg/ml) at 37°C. After

746

3 h of growth (at an O.D. of 0.8), cultures were induced with 1 mM isopropylthio-S-Dgalactoside (IPTG) and continued to grow for different durations; cells were stored at 4°C at the end of the time course. SDS-gel electrophoresis SDS-polyacrylamide gels were prepared and electrophoresed according to Laemmli (1970). From 1.5 ml culture, cells were pelleted by centrifugation, washed once in 500 ~1 of Tris.Cl(50 mM, pH 7.6) and resuspendedin 100 ~1 of distilled water. From this, 20 ~1 sample was boiled with an equal volume of 2x SDS gel loading buffer (100 mM Tris.Cl-pH 6.8, 200 mM dithiothreitol, 4 % SDS, 0.2% bromophenol blue and 20% glycerol) for 5 minutes. After electrophoresis, polypeptides were visualized by negative staining with CuCl, (Lee et al., 1987). Transmission electron microscopy Cells were pre-fixed in 3% glutaraldehyde and post-fixed in 1% osmium tetroxide (final concentrations) in 0.05M cacodylate buffer followed by a buffer wash to remove any unbound osmium. Pellets were solidified with 2% agarose and minced into 1 mm3 size blocks, dehydrated in a graded series of ethanol followed by propylene oxide treatment and embedded in Spur’s resin (Spurr, 1969). Sections were stained with uranyl acetate (1%, pH-4.0) for 40 minutes and lead citrate @H- 12.0) for 2 minutes. Specimens were observed under a Zeiss transmission electron microscope. RESULTS AND DISCUSSION Cell lysates of both TB and LB grown cultures separatedon SDS-polyacrylamide gels are shown in Figure 1. Polymer protein can be seen by negative staining around 60 kDa (Fig. 1). The pattern of polymer production is observed to be the same in both gels, although the quantity of polymer is several fold more in TB grown cultures (uninduced). The amount of polymer in uninduced 6 h sample (lane 3) is approximately comparable to that of the induced 6 h sample (lane 4). However, there is a dramatic increase in the expression of polymer in uninduced cultures grown for 24 h (lane 5) over induced cultures of the sameage (lane 6). This increase is more pronounced in TB grown cultures compared to LB grown cultures which is not surprising becauseit is known that in TB grown cultures, copy number of the plasmid increases by 4-7 fold and the cell density increasesby 10 fold over those of the LB grown cultures (Tartof and Hobbs, 1987). In contrast, the amount of polymer produced in induced cultures is negligible (lanes 6, 8 and 10) accompaniedby irregular shapesof cells (see Fig. 2D). Decrease in polymer production in induced cells could be directly correlated with loss of the introduced plasmid and reduced cell growth. No plasmid DNA was found in cells induced with IPTG beyond 6 h of growth (data not shown). Such reduction in cell growth after IPTG induction has been observed earlier (Brosius, 1984; Masui et al., 1984). In our studies, the highest expression

F’igure 1. Crude protein extractsfrom E. coli strain HMS 174 (DE3) transformedwith PET lld-120mer separatedon SDS-PAGE gels. A. Cultures grown in Terrific Broth (TB). B. Cultures grown in Luria Broth (LB). Lanes 1. High range protein marker showing (Top to bottom) myosin, S-galactosidase, phosphorylaseb, bovine serum albumin and ovalbumin; 2. Partially purified polymer standard; 3. Uninduced-6 h; 4. Induced-6 h; 5. Uninduced-24 h; 6. Induced-24 h; 7. Uninduced-48 h; 8. Induced-48 h; 9. Uninduced-72 h; 10. Induced-72 h; 11. Host strain without plasmid; 12. Uninduced-48 h culture stored for 3 months at 4°C. For induction 1mM IPTG was added when the cultures reached 0.8 O.D.

of protein is observed in 24 h uninduced cultures (lane 5), followed by a gradual reduction in cultures grown beyond 24 h (lanes 7 and 9). This may be due to cell lysis as well as decrease in plasmid copy number after 24 h as evident from light microscopic observations and plasmid DNA isolation studies (data not shown). Synthesizedpolymer is extraordinarily stable in E. coli cells as seen in Figure 1 (lane 12) that shows polymer isolated from a 48 h uninduced culture stored for more than three months at 4°C. The polypeptide observedat the samemolecular weight as B-galactosidase(116.3 kDa), after 24 h of growth (lanes 6, 8, 10) in induced cells but not in uninduced cells (lanes 3, 5, 7, 9) may be l3-galactosidase(chromosomalgene) induced by addition of gratuitous inducer (IPTG). This result is in accordancewith earlier reports that bacteria produce 13-galactosidase only when its substrateis added to the medium (Lewin, 1990). Excessproduction of polymer in 24 h old uninduced cells may also be attributed to reduction or dilution of repressorprotein as evidenced by increase in D-galactosidaseproduction in induced cells of the sameage (Fig. 1). Dilution of the repressor protein in rapidly growing cells should have enabled the RNA polymerase to

748

efficiently bind and initiate transcription of the T7 promoter that drives the polymer gene. It is known that the amount of repressor protein produced by DE3 on host chromosome is not sufficient to block the T7 polymerase transcription; BL21 (DE3) cells transformed with the pT75 plasmid that carries a cryZZ4 gene driven by the T7 promoter produced large quantities of @IA

crystals without any need for induction with IPTG (Daniel1 et al., 1994).

Figure 2. Transmission electron micrographsof E. coli strain HMS 174 (DE3) transformed with pETlId120mer showing polymer production at different durations of culture growth in uninduced and induced cells. A. Uninduced-6 h ; B. Induced-6 h; C & E. Uninduced-24 h; D. Induced-24 h; F. Host cell without the pETlId120mer plasmid.

Electron micrographs obtained at different stagesof growth of induced and uninduced cultures are shown in Figure 2. At 6 h, uninduced cells are rod shaped with smooth cell wall and several inclusion bodies all along the cell (Fig. 2A). But induced cells appear deformed with an irregular cell wall and poorly defined inclusion body (Fig. 2B). As growth progressed upto 24 h, uninduced cells show a dramatic increase in the number and size of inclusion bodies pushing the cytoplasm aside and occupying the cell volume to a maximum extent possible (Fig. 2C, 2E). Polymer inclusions appear as glittering bodies amidst a dense dark background of the cell cytoplasm; these structues are distinct from the cell cytoplasm by their lighter stain, round to oval shape with poorly infiltrated regions showing dense reflecting matrix. In some cells

749

there are single large inclusion bodies occupying about 7080% of the cell volume (Fig. 2E) whereas in other cells fewer bodies occupy nearly 80-90% of the cell volume (Fig. 2C). In contrast, induced cells have irregular cell wall with dense cytoplasm and no inclusion body (Fig. 2D). Host cells not transformed with plasmid DNA show normal cell growth with no inclusion body (Fig. 2F). Quantitative analyses of electron micrographs using BioScan Gptimas Version 3.10 revealed the volume occupied by these bodies to be as much as 80-90% of the cell volume under optimal growth conditions. The mean area occupied by the polymer in fully grown cells was 6575% of the cell volume. Inclusion bodies generally are formed when proteins are synthesizedto levels above their solubility. ACKNOWLEDGMENT

This work was supported in part by the Office of Naval Research grant # NOO14-89-J-1970 (DWU), by the U. S. Army, Natick Research contract # DAAK60-93-C-0094 (DWU) and by the NIH grant # GM 16551-01 (HD). The authors are thankful to Drs. R.R. Dute and W.J. Moar of Auburn University for critical discussions. REFERENCES Brosius, J. (1984). Gene 27, 161-172. Daniell, H., PoroboDessai, A., Prakash, C.S. and Moar, W.J. (1994). In: Biochemicul and Cellular Mechanisms of Stress Tolerance in Plants, J.H. Cherry, ed. vol. H 86, pp. 589604, NATO AS1 Series in Cell Biology, Springer-Verlag, Berlin Heidelberg. Krejchi, M-T., Atkins, E.D.T., Waddon, A.J., Foumier, M.J., Mason, T.L. and Tirrell, D.A. (1994). Science 265, 1427-1432. Laemmli, U.K. (1970). Nature 227, 680-685. Lee, C., Levin, A. and Branton, D. (1987). Anal. Biochem. 166, 308-312. Lewin, B. (1990). Genes IV, pp. 240. Oxford University Press, New York. Masui, Y., Mizuno, T. and Inouye, M. (1984). Bio/TechnoZogy 2, 81-85. McPherson, D.T., Morrow, C., Mineham, D.J., Wu, J., Hunter, E. and Urry, D. W. (1992). Biotechnol. Prog. 8, 347-352. Spurr, A.R. (1969). J. Ultrastruct. Res. 26, 31-36. Studier, F.W. and Moffat, B.A. (1986). J. Mol. Biol. 189, 113-130. Tartof, K.D. and Hobbs, C.A. (1987). Bethesda Res. Lab. Focus 9, 12. Urry, D.W. (1995) Scientific American 272, 64-69. Urry, D.W., McPherson, D.T., Xu, J., Daniell, H., Guda, C., Gowda, D.C., Jing, N. and Parker, T. M. (1994). In: The Polymeric Materials Encyclopedia: Synthesis, Properties and Applications, J.C. Salamone, ed. pp. 2645-2699, CRC Press, Boca Raton. Urry, D.W., Nicol, A., Gowda, D.C., Hoban, L.D., McKee, A., Williams, T., Olsen, D.B. and Cox, B.A. (1993). In: Biotechnological Polymers: Medical, Pharmaceutical and Industrial Applications, C.G. Gebelein, ed. Technomic Publishing Co., Inc., Atlanta, GA. Urry, D. W., Parker, T.M., Reid, M.C. and Gowda, D. C. (1991). J. Bioactive Compatible Polym. 6, 263-282.

750