Manipulation and expression of the maize zein storage proteins in Escherichia coli

Jo,m,al of Biotechnology, 2 (1985) 157-175

157

Elsevier JBT 00145

Manipulation and expression of the maize zein storage proteins in Escherichia coli Jan M. N o r r a n d e r 1.,, Jeffrey Vieira 1, Irwin Rubenstein 2 and J o a c h i m Messing 1 Departments of t Biochemistry, and : Genetics and Cell Biology, University of Mhmesota, St. Paul. MN 55108. U.S.A.

(Received 30 November1984; accepted3 December1984)

Summa~ The cDNA sequence for the mature form of the zein protein A20 was inserted into the lac cloning region of two different M13mp phage vectors. Translation of these recombinant phage in E. coli cells produced a fl-galactosidase-zein fusion protein. Another zein clone was constructed in which the entire coding sequence, including that of the signal peptide, was cloned into a M13mp vector. This clone was designed to produce a pre-zein protein which did not contain any fl-galactosidase amino acids. Expression of these phage-coded proteins in E. coli cells was detected on Western blots using zein antibodies. Expression of the fl-galactosidase-zein fusion protein was extremely low, comprising less than 1% of the total E. coli proteins. Levels of expression were increased slightly when this same sequence was cloned into a pUC-derived expression plasmid containing the highly efficient t r p - l a c promoter. oligonucleotide-directed in vitro mutagenesis, M13mp and pUC vectors, protein engineering

Introduction The zeins are a group of related proteins coded for by a multigene family. They are the major storage proteins of corn, constituting more than 50% of the protein in the mature endosperm (Nelson, 1966). They are found packaged in membrane-bound * Present address: AMGen, Inc., ThousandOaks, CA 91320-1789, U.S.A.

0168-1656/85/$03.30 © 1985 ElsevierSciencePublishers B.V.(BiomedicalDivision)

158 organelles called protein bodies (Duvick, 1961; Wolf et al., 1967; Christianson et al., 1968). Zeins are synthesized on membrane-bound polysomes and contain 20-21 amino acid signal peptides on their amino terminus. These pre-zein proteins are processed to mature zein proteins by cleavage of the signal peptide as the protein passes through the protein body membrane (Larkins et al., 1979; Burr and Burr, 1981). Zein proteins are important in mammalian nutrition due to the widespread use of corn as a food source. However, the zein proteins contain very small amounts of some essential amino acids, specifically lysine and tryptophan (Wall, 1964), which limits the nutritional value of corn. If these amino acids could be added to the zein proteins, the nutritional value of corn would be greatly increased. Therefore, zein proteins are attractive candidates for genetic engineering aimed at improving their value as a food source (Messing, 1983b). Oligonucleotide-directed in vitro mutagenesis is a technique which uses synthetic oligodeoxyribonucleotides to make specific changes in a sequence of DNA (Hutchinson et al., 1978). This technique has been used to create point mutations, deletions and insertions. The single-stranded M13mp phage vectors are versatile tools for this type of mutagenesis (Zoller and Smith, 1982; Norrander et al., 1983). They enable a protein-coding sequence to be cloned, sequenced, mutated and expressed all in the same vector. A protein coding sequence inserted under the control of the M13mp lac promoter will be expressed in infected cells. High levels of expression, up to 5070 of total soluble proteins, have been reported using M13mp phage as expression vectors (Winter et al., 1982; Slocombe et al., 1982). Oligonucleotide-directed in vitro mutagenesis and M13mp phage vectors have been used in the study of protein structures and function (Dalbadie-McFarland et al., 1982; Villafranca et al., 1983; Wilkinson et al., 1983). Alterations made in a gene sequence using a synthetic oligonucleotide will be reflected in the sequence of the gene product, resulting in the production of a mutant protein. These mutant proteins can be studied to determine the effects of the sequence changes on their structure and function. These techniques may also be applied to the engineering of new and more versatile proteins by incorporating changes in a gene sequence designed to improve the usefulness of the resulting protein product. In the work presented here, oligonucleotide-directed in vitro mutagenesis techniques were applied to the construction of recombinant M13 phage vectors containing the coding sequences for zein proteins. In contrast to other foreign proteins, zein proteins are poorly expressed in E. coli, even when the coding cartridge is transferred to a pUC-derived expression plasmid containing the highly efficient trp-lac promoter. The construction design, however, makes it easy to manipulate the coding sequence and transfer the altered codon-cartridge to a plant shuttle vector. Materials and Methods

Strains E. coil JMI09 cells were used (Yanisch-Perron et al., 1984). Cells were maintained, cultures grown and transfection and transformations carried out as described by Messing (1983a).

159

Preparation, sequencing and cloning of DNA Single- and double-stranded M13mp phage DNAs and pUC plasmid DNAs were prepared as described by Messing (1983a). Dideoxy sequencing and cloning procedures were carried out as described by Messing (1983a). Oligodeoxyribonucleotide synthes& The oligodeoxynucleotides used in this work were synthesized using the phosphoramidite method (Beaucage and Caruthers, 1981) on a solid-phase automated DNA synthesizer from Systec, Inc. (Minneapolis, MN, U.S.A.). Oligodeoxyribonucleotides were used directly after removal from the support without further purification. Oligonucleotide-directed in oitro mutagenesis Oligonucleotide-directed in vitro mutagenesis was carried out as described by Zoller and Smith (1982). Plaque lifts were carried out as described by Norrander et al. (1983). Production of zein in infected E. coli cells Exponentially growing E. coli JM109 cells were diluted to a concentration of 8 × 106 cells per ml and infected with a 1 : 1 ratio of M13mpl0/A20, M13mp321/A20 or M13mp32N/ZG7 phage particles. Uninfected JM109 cells and M13mp321-infected cultures were grown as controls. The cultures were incubated at 37°C for 2 h. The cultures were then divided into two equal parts; IPTG (0.5 mM) was added to one and incubation was continued at 37°C. 1 ml samples were removed and zein isolated as described below. Production of zein in transformed E. coli cells Cultures of 2XYT broth (Miller, 1972), containing 100 /~g ml -~ of ampicillin, were inoculated with single colonies of JM109 cells transformed with p U C 1 2 / A 2 0 or pUC14tac/A20 and incubated at 30°C. Control cultures containing untransformed JM109 cells and JM109 cells transformed with pUC18 or pUC14tac were also started. When the cells reached a concentration of 2 × 10 s cells per ml, the cultures were transferred to 37°C and incubated for 2 h. The cultures were then divided into two equal parts; IPTG (0.5 mM) was added to one and incubation was continued at 37°C. Samples of 1 ml were removed and zein isolated as below. Isolation of zein from E. coli cells The cells in 1 ml of an infected or transformed cell culture were pelleted by centrifugation at 7500 rpm for 5 min in a Beckman JS-7.5 rotor. The cells were washed in 0.5 ml of water and repelleted by centrifugation. The cells were resuspended in 0.5 ml of 10% TCA and sonicated for 5 s. The samples were incubated on ice for 30 min. Precipitated proteins were pelleted by centrifugation at 15 000 rpm for 15 min in a Beckman JA-17 rotor. The protein pellet was washed three times in 0.5 ml of water to remove residual TCA and the proteins repelleted by centrifugation. The protein pellet was resuspended in 0.25 ml of a solution containing 70%

160 ethanol, 1 mM PMSF and 17o 2-mercaptoethanol. The solution was boiled for 5 min, vortexed for 30 s and centrifuged at 15000 rpm for 15 min in a Beckman JA-17 rotor. The supernatant fraction, containing the zein protein, was removed and dried down in a Speed-Vat (Savant Instruments, Inc.). The dried protein was resuspended in 20 /~I of a solution containing 0.017o bromphenol blue, 57o glycerol, 5,% 2mercaptoethanol, 270 SDS and 30 mM Tris-HC1 (pH 6.8). The samples were boiled for 5 rain, vortexed for 30 s and then centrifuged for 5 min in an Eppendorf centrifuge to pellet any undissolved protein. The samples were loaded on SDS-polyacrylamide gels as described below.

Isolation of zein from culture media The cells in 1 ml of a culture were pelleted by centrifugation at 7500 rpm for 5 min in a Beckman JS-7.5 rotor; next, 0.25 ml of 407o TCA were added to the supernatant fraction and the solution was incubated on ice for 30 min. Precipitated proteins were pelleted by centrifugation at 15000 rpm for 15 min in a Beckman JA-17 rotor. The resulting protein pellet was washed, ethanol-extracted and prepared for SDS-polyacrylamide gel electrophoresis as described above.

SDS-polyacrylamide gels Protein isolated from E. coli cells were separated by electrophoresis on SDS-polyacrylamide stacking gels using a Hoefer vertical slab gel apparatus (Laemmli, 1970; Lee et al., 1976; Burr et al., 1978). After electrophoresis, the separated proteins were either transferred to nitrocellulose by the Western procedure described below or visualized by staining with Coomassie Brilliant Blue R.

Western transfer 1. Transfer of protein to nitrocellulose. Proteins were transferred from SDS-polyacrylamide gels to nitrocellulose using a Hoefer electroblotting apparatus (Towbin et al., 1979). After the electrophoresis was completed, the SDS gels were incubated for 30 min at 23°C in elution buffer containing 25 mM Tris base, 192 mM glycine and 2070 (v/v) ethanol to remove the SDS from the gel. A sheet of nitrocellulose, cut to the size of the gel, was soaked in water for 15 min. Four sheets of gel size Whatman 3MM filter paper, prewet in elution buffer, were placed on the anode side of the electroblotter sandwich apparatus. The gel was floated onto the wet nitrocellulose sheet and then placed on top of the four sheets of filter paper, with the nitrocellulose sheet on the anode side and the gel on the cathode side of the sandwich. Another sheet of Whatman 3MM filter paper, prewet in elution buffer, was placed on top of the gel. Any air bubbles present were removed. The sandwich was placed in the electroblotting apparatus with the gel on the cathode side and nitrocellulose on the anode side. Electrophoresis was carried out at 0.1 A (45-50 V) for 2 h using elution buffer which had been pre-cooled to 4°C. 2. Prehybridization. The nitrocellulose filter was placed in a heat-sealable bag with 10 ml of a solution containing 57o BSA/0.9% NaC1/10 mM Tris-HCl (pH 7.4) and incubated at 37°C for 3 h.

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3. Hybridization of zein ant&erum. The prehybridization solution was removed from the bag and replaced with 10 ml of a 1 : 50 dilution of zein antiserum in 5% BSA/0.9% NaCI/10 mM Tris-HCl (pH 7.4) and incubated at 23°C for 3 h. The filter was removed from the bag and washed 5 × in 100 ml of 0.9% NaC1/10 mM Tris-HCl (pH 7.4) at 23°C for a total of 30 min. The zein antiserum was prepared as described in Matzke et al. (1984). 4. Hybridization of peroxidase-conjugated antibody. The nitrocellulose filter was placed in a heat-sealable bag with 10 ml of a 1 : 2000 dilution of peroxidase-conjugated anti-rabbit IgG (Cappel Lab #3212-0084) in 5% BSA/0.9% NaC1/10 mM Tris-HCi (pH 7.4) and incubated at 23°C for 2 h. The filter was removed from the bag and washed 5 x in 100 ml of 0.9% NaC1/10 mM Tris-HC1 (pH 7.4) at 23°C for a total of 30 min. 5. Reaction of peroxidase with o-dianisidine. The nitrocellulose filter was incubated in 250 ml of a solution containing 25 /tl m1-1 o-dianisidine in 0.01% H2OJ10 mM Tris-HC1 (pH 7.4) for 30 min at 23°C. The reaction was stopped by incubating the nitrocellulose filter in water for 5 min at 23°C. The filter was air dried and stored for protection from light.

Results

Construction of M13mp10 / A20 The DNA sequence coding for the mature form of the zein protein A20 was cloned into the phage vector M13mpl0 as diagrammed in Fig. 1. A cDNA clone for the A20 protein was first constructed by Burr et al. (1982) in the plasmid pMB9. The DNA sequence of this clone was determined by Geraghty et al. (1982) and found to contain 110 base pairs of 5' leader sequence, 721 base pairs of coding sequence and 89 base pairs of 3' nontranslated sequence. The coding sequence for the mature form of the protein was transferred to the plasmid pUC13 to form the clone pUC13/A20. The plasmid pUC13/A20 was constructed by cloning the FnuDII-HindlII fragment from the cDNA clone A20 into the EcoRI-HindlII sites of the plasmid pUC13. The enzyme HindlII cuts the pMB9 plasmid 220 base pairs after the 3' end of the A20 cDNA insert. The enzyme FnuDII makes a blunt cut in the A20 sequence two base pairs before the first codon of the mature zein protein. When this fragment was cloned into the cut and filled in EcoRI site of the plasmid pUC13, the EcoRI site was regenerated. This made it possible to reisolate a fragment containing the coding sequence for the mature form of the zein protein A20 by digestion of pUC13/A20 with EcoRI and HindlII. The EcoRI-HindlII fragment from pUC13/A20 was cloned into the EcoRI-HindlII sites of the phage vector M13mpl0 to produce the phage M13mpl0/A20. The coding sequence for the mature A20 protein is under the control of the lac promoter as shown in Fig. 2. The codons for the first six amino acids of the protein /3-galactosidase are present 5' of the A20 coding sequence. Translation of this sequence in infected E. coli cells produces a/3-galactosidase-zein

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fusion protein containing the first six amino acids of/3-galactosidase at the amino terminus of the mature A20 zein protein.

Construction of M13mp321 / A 20 The sequence coding for the mature form of the zein protein A20 was also cloned into the phage vector M13mp321 to form the clone M13mp321/A20. Translation of this phage clone produces the same/~-galactosidase-zein fusion protein as the clone M13mpl0/A20. However, the construction of the phage M13mp321 gives it advantages over other M13mp phage vectors for use as an expression vector. M13mp321, diagrammed in Fig. 3, differs from other M13mp phage vectors in

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three ways (Vieira and Messing, in prep.). First, M13mp321 contains a smaller portion of the lac operon compared to other M13mp phage, consisting of the HaelI fragment contained in the pUC plasmids (Vieira and Messing, 1982). Second, this lac fragment was inserted into a different site in the M13 molecule. Other M13mp phage, such as M13mpl0, have the lac region inserted as described by Messing et al. (1977). In M13mp321, the lac region was inserted into the HaelI sites at positions 5559 and 5567 on the M13 map. Third, the HaelI lac fragment is inserted in the opposite orientation as compared to other M13mp vectors. As a result, in M13mp321 the coding strand of the lac operon is in the negative phage strand. In other M13mp vectors, the positive strand found in the phage particles contains the coding strand of the lac operon. The reverse orientation of the lac fragment is advantageous when the M13mp phage are used as expression vectors. For expression to occur, the coding sequence of the desired protein must be inserted into the M13mp vector in frame with the first

164

ATG of the lac z gene. To verify the frame of the insert, the region coding for the amino terminus of the protein must be sequenced. This is difficult to do in M13mpl0/A20. To sequence this region would require sequencing through the entire protein coding sequence, starting at the carboxyl terminus coding region, and any 3'-nontranslated sequence before the desired amino terminus coding region was reached (Fig. 2). For inserts over 600 base pairs it would be impossible to sequence this region using the universal sequencing primer. New primers, homologous to the protein coding sequence, would have to be designed for each new protein insert. However, using the phage M13mp321 the same primer can be used to sequence the amino terminus coding region of any protein sequence cloned into the polylinker region. This primer, called the reverse primer, hybridized to the M13mp321 template DNA (+ strand) as diagrammed in Fig. 3 and allows the determination of the insert starting at the amino terminus coding region.

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165

Construction of M13mp32N / ZG7 To compare the expression in E. coli cells of zein fusion proteins - the fusion between the amino terminus of the fl-galactosidase and the mature zein protein with a non-fusion protein, a sequence coding for an entire zein protein, including the signal peptide, was cloned into the phage vector M13mp32N. The vector M13mp32N differs from the phage M13mp321 in that it contains an NcoI site (GGATCC) at the initiation codon of the fl-galactosidase coding sequence (Fig. 3). As a result, a protein coding sequence which contains an NcoI recognition sequence at its initiator codon can be inserted in frame with the initiator codon of the lac z gene by cloning into the NcoI site of M13mp32N. The resulting translational product will be an authentic protein and will contain no amino acids from fl-galactosidase. If an NcoI site is not present at the initiation codon of a protein sequence, one can be introduced using oligonucleotide-directed in vitro mutagenesis. A new 18 base primer (GTTGTGTGGAATTGTGAG), reverse primer-18 (RP18), was designed for use with the M13mp32N phage vector (Fig. 3). This primer hybridizes 40 bases 5' of the reverse primer used with M13mp321 and therefore enables sequence to be read starting at the NcoI site of M13mp32N. The amino terminus coding sequence of a protein cloned into the NcoI site of M13mp32N can be sequenced using this primer to make sure it is in frame with the ATG of the lac z gene. Several attempts were made to introduce an NcoI recognition sequence into a recombinant phage constructed by inserting the A20 containing H i n d l I I - S a l I fragment of pMB9/A20 into M13mp32N. No peaks corresponding to doublestranded, covalently-closed molecules were detected when mutagenesis reaction mixtures were run on alkaline sucrose gradients. This may have been due to secondary structures within the A20 or pMB9 sequences which prevented the completion of the second strand of the phage molecule. To overcome this problem, the procedure diagrammed in Fig. 4 was carried out using the zein clone pZG7. pZG7 is a zein cDNA sequence cloned into the plasmid pUC9 (Heidecker and Messing, 1983). This zein clone contains a unique BamHI site 170 base pairs from its initiator codon and is flanked by the HindlII and SalI sites of the pUC9' polylinker region. Digestion of pZG7 with HindlII and BamHI produces a 240 base-pair fragment containing the initiation codon for the ZG7 coding sequence. This fragment was cloned into the phage vector M13mpl8 (M13mpl8/HB-pZG7), The sequence flanking the initiation codon of ZG7 was then altered to create the recognition site for the restriction enzyme NcoI. The resulting phage containing the NcoI recognition sequence was named M13mpl8/HB-pZG7-N. This sequence change was accomplished by using a 16-mer to change one base pair immediately flanking the initiation codon in the 5'-noncoding region of the pZG7 sequence. The amino acid sequence of the ZG7 protein was not altered by this mutation. The mutagenesis was carried out as described in Materials and Methods. Three out of 74 plaques, or 4%, remained hybridized to the mutant primer after a stringent wash at 42°C. The B a m H I - S a l I fragment of pZG7, containing the rest of the ZG7 coding sequence, was cloned into the phage vector M13mp32N to form the recombinant phage M13mp32N/BS-pZG7 (Fig. 4). The ZG7 coding sequence was restored by

166

transferring the N c o I - B a m H I fragment from M13mpl8/HB-pZG7-N to M13mp32N/BS-pZG7 to form the clone M13mp32N/ZG7 (Fig. 4). To verify that the ZG7 coding sequence was in frame with the ATG of the lac z gene, M13mp32N/ZG7 template DNA was isolated and sequenced using the reverse primer-18 (RP-18). When translated this phage will produce a zein protein containing its signal peptide as diagrammed in Fig. 5. Unlike the recombinant phage M13mpl0/A20 and M13mp321/A20, the protein translated from M13mp32N/ZG7 will not contain any amino acids from fl-galactosidase. HoelI l-.loe IT A

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ZG7 coding sequence, was cloned into the BamHI-SalI sites of M13mp32N to form the phage M13mp32N/BS-pZG7 (left column). The entire coding sequence of ZG7 was then restored by inserting the NcoI-BamHI fragment from M13mplS/HB-pZG7-N into the NcoI-BamHI sites of M13mp32N/BS-pZG7 to form the phage M13mp32N/ZG7.

167

Construction of pUCl2 / A 2 0 and pUCl4tac/A20 The coding sequence for the mature form of the protein A20 was cloned into the plasmids pUC12 (Vieira and Messing, 1982) and pUC14tac, in an attempt to achieve higher levels of expression, pUC14tac is a pUC derived expression plasmid which contains the high expression trp-lac promoter (Amann et al., 1983) in place of the lac promoter found in pUC12 (Vieira and Messing, in prep.). A 10-fold higher level of transcription is seen using the trp-lac promoter as compared to the lac promoter (Amann et al., 1983). Both plasmids are high copy number plasmids, producing approximately 200 copies per cell, and have the same polylinker arrangement as the M13mp phage counterparts (Vieira and Messing, in prep.). This high copy number, along with the high expression prooaoter in pUC14tac, should produce increased levels of zein in E. coil cells transformed with pUC12/A20 or p U C 1 4 T / A 2 0 as compared to E. coil cells transfected with M 1 3 m p l 0 / A 2 0 or M13mp321/A20. Translation of p U C 1 2 / A 2 0 or pUC14tac/A20 in E. coli cells produces the same fl-galactosidase-zein fusion protein as the recombinant phage M 1 3 m p l 0 / A 2 0 and M13mp321/A20. Production of zein in E. coil cells infected with M13mplO/A20, M13mp321/A20 and M13mp32N/ZG7 E. coli cells were infected with these recombinant phage and zein isolated as described in the Materials and Methods. As controls, the isolation procedure was

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also carried out on uninfected E. coli JM109 cells and M13mp321-infected JM109 cells. It was not possible to detect the production of the fl-galactosidase-zein fusion protein or pre-zein protein coded for by these phage on SDS-polyacrylamide gels due to the numerous E. coli proteins which are visible, some of which comigrate with the major zein proteins. However, when a Western transfer was carried out, the production of zein was easily detected in E. coil cells infected with M13mp10/A20 and M13mp321/A20 (Fig. 6, lanes 5-8). An increased level of production was detected when IPTG was added to the cell cultures. Both of these phage produced proteins which comigrated with the 19000 dalton zein protein. This is the size predicted from the A20 protein sequence (Geraghty et al., 1982). There are faint bands visible just below the 19000 dalton band in the extracts from cells infected with M13mp10/A20 or M13mp321/A20 and induced with IPTG. These bands

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22,500 19,000

Fig. 6. Immunodetection of zein proteins isolated from E. coil cells infected with M 1 3 m p l 0 / A 2 0 , M 1 3 m p 3 2 1 / A 2 0 and M 1 3 m p 3 2 N / Z G 7 . JM109 cells were infected with the recombinant phage M 1 3 m p l 0 / A 2 0 , M13mp321/A20 and M 1 3 m p 3 2 N / Z G 7 . Cultures were grown up with and without induction by IPTG and zein was isolated as described in the Materials and Methods. Controls with uninfected and M13mp321-infected cells were also carried out. The resulting protein fractions were subjected to electrophoresis on a SDS-polyacrylamide gel, transferred to nitrocellulose paper and hybridized to zein antiserum. Lane 1 = 5 p.g of zein proteins; Lane 2 = uninfected cells; Lane 3 = uninduced M13mp321-infected cells; Lane 4 = induced M13mp321-infected cells; Lane 5 = uninduced M13mpl0/A20-infected cells; Lane 6 = induced M13mpl0/A20-infected cells; Lane 7 = uninduced M13mp321/A20-infected cells; Lane 8 = induced M13mp321/A20-infected cells; Lane 9 = uninduced M13mp32N/ZG7-infected cells; Lane 10 = induced M13mp32N/ZG7-infected cells.

169 possibly arose from some proteolytic cleavage of the fl-galactosidase-zein fusion proteins in the E. coli cells. The level of production of zein by these two recombinant phages was low compared with other reports of expression using M13mp vectors (Winter et al., 1982; Slocombe et al., 1982). There appears to be less than 1.0/~g per ml of cell culture compared to a standard with purified zein. This is less than 1% of the total E. coli proteins compared to 50% obtained by Winter et al. (1982). Initially, zein could not be detected in cells infected with the phage M13mp32N/ZG7 (Fig. 6, lanes 9-10). Since this clone differed from M13mpl0/A20 and M13mp321/A20 in containing the sequence coding for the signal peptide, it was possible that this protein was being exported from the E. coli cells into the culture media. To check for export of this protein as well as the fl-galactosidase-zein fusion protein produced by M13mpl0/A20 and M13mp321/A20, ethanol-soluble proteins were isolated from 1 ml of culture media as described in the Materials and Methods. The isolated proteins were subjected to electrophoresis on SDS-polyacrylamide gels and a Western transfer carried out. No zein proteins were detected in the culture media from any of the recombinant phage. Another possible reason for the failure to detect any synthesis of zein in cells infected with M13mp32N/ZG7 was that some mutation had occurred in the promoter or other regulatory region of the phage molecule. Since the M13mp32N/ZG7 phage supernatant used to infect the E. coli cells was prepared from a single plaque, it was possible that supernatant isolated from other M13mp32N/ZG7 phage plaques may not contain the mutation and therefore produce zein. Phage supernatant from the original M13mp32N/ZG7 isolate (No. 59) and a second isolate (No. 116) shown to be identical by restriction ana]ysis was streaked for single plaques on B-broth plates. Phage supernatant was prepared from 4 of the resulting plaques from each of the clones. These phage supernatants were used to infect cultures of E. coli cells. The original 59 and 116 phage supernatants were also used to infect E. coli cells. The zein isolation procedure was carried out on these infected cells. The resulting ethanol-soluble protein was subjected to electrophoresis on a SDS-polyacrylamide gel and a Western transfer carried out. Two faint bands appeared in some of the lanes. The lower molecular weight band migrates at approximately the same place as the 19000 dalton zein protein (Fig. 7). The upper band may be the unprocessed form of the ZG7 protein and the lower band the processed form. If these bands do represent the processed and unprocessed forms of the ZG7 proteins, expression of this protein is extremely low and in fact undetectable in some isolations. Production of zein in E. coli cells transformed with p UC12 / A20 and p UCl 4tac / A20 E. coil cells were transformed with pUC12/A20 or pUC14tac/A20 and single colonies grown up as described in the Materials and Methods. Ethanol-soluble proteins were isolated and subjected to electrophoresis on SDS-polyacrylamide gels. Controls were carried out using untransformed JM109 cells and JM109 cells transformed with pUC18 or pUC14tac. The resulting SDS-polyacrylamide gel is shown in Fig. 8. No expression of zein was detected in cells transformed with the plasmid pUC12/A20 either with or without the addition of IPTG. Very few E. coli

170

proteins were also isolated from these samples compared to the untransformed and pUC18-transformed controls, indicating a lower concentration of cells was present in the pUC12/A20-transformed culture. The concentration of all the cell cultures was approximately 1 x 109 when the IPTG was added. Therefore, a decrease in the growth rate of the pUC12/A20-transformed cells must have occurred after this point. The reason for this decreased growth is unknown. Cells transformed with the plasmid pUC14T/A20 produced the highest levels of zein (Fig. 8, lanes 9-10). There was no difference in the expression levels seen in induced and uninduced cell cultures. This lack of repression in uninduced cells may be due to the high copy number of the pUC14T plasmid. As with the pUC12/A20-transformed cells, a decrease in the total number of proteins isolated is seen in pUC14T and pUC14T/A20-transformed cells as compared to the untransformed and pUC18-transformed controls. Since cells transformed with pUC14T show a decreased rate of growth, a lower concentration of cells in the pUC14T- and pUC14T/A20-transformed cultures probably accounts for the decreased amount of total protein isolated. The difference seen between induced and uninduced

1

2

3

4

5

6

7

8

9

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22,500__ 19,000--

Fig. 7. Immunodetection of zein proteins isolated from M13mp32N/ZG7-infected E. coli cells. Phage supernatants from two separate isolates of the recombinant phage M13mp32N/ZG7 (59 and 116) were streaked on B-broth plates. Single plaques were picked from each and phage supernatant prepared. E. coil JM109 cells were infected with the original isolates (59 and 116) and the phage supernatants prepared from each (59-1, 59-2, 59-3, 59.4, 116.1, 116.2 and 116-3). All cultures were induced with 1PTG and ethanol.soluble proteins isolated as described in the Materials and Methods. The resulting protein fractions were subjected to electrophoresis on a SDS-polyacrylamide gel, transferred to nitrocellulose paper and hybridized to zein antiserum. Lane 1 - 5/~g of zein proteins; Lane 2 - 59; Lane 3 - 59-1; Lane 4 - 59-2; Lane 5 - 59-3; Lane 6 - 59-4; Lane 7 - 116; Lane 8 - 116-1; Lane 9 - 116-2; Lane 10 - 116-3.

171

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22,500_ 19,000--

.4""

Fig. 8. Zein proteins isolated from E. coil cells transformed with pUC12/A20 and pUC14tac/A20: SDS-polyacrylamide gel. E. coli JMI09 cells were transformed with the plasmids pUC12/A20 and pUCl4tac/A20. Cultures were grown up with and without induction by IPTG. Zein was isolated as described in the Materials and Methods. Controls of untransformed, pUCl8-transformed and pUC14tactransformed cells were also carried out. An SDS-polyacrylamide gel of the resulting ethanol soluble fractions is shown. Lane 1 - 5 #g of zein proteins; Lane 2 - untransformed cells; Lane 3 - uninduced pUCl8-transformed cells; Lane 4 - induced pUC18-transformed cells; Lane 5 - uninduced pUCl2/A20-transformed cells; Lane 6 - induced pUCl2/A20-transformed cells; Lane 7 - uninduced pUCl4tac-transformed cells; Lane 8 = induced pTLl4-transformed cells; Lane 9 - uninduced pUC14tac/A20.transformed cells; Lane 10 - induced pTLl4/A20-transformed cells.

pUC14tac-transformed cells indicated that growth may be slowed even more after the addition of IPTG. The combined effect of a high expression of the pUC14tac/A20-coded/~-galactosidase-zein fusion protein and a low background of E. coli protein made it possible to easily detect the production of this zein directly on a SDS-polyacrylamide gel (Fig. 8, lanes 9-10).

Discussion

The use of oligonucleotide-directed in vitro mutagenesis to alter a protein coding sequence has important applications to the study of proteins. One such application would be the addition of codons to a gene sequence which code for specific amino acids designed to improve the usefulness of the resulting protein product. One group of proteins which could benefit from such alterations are the zein storage proteins of corn. The zein proteins contain very small amounts of some essential amino acids,

172 specifically lysine and tryptophan, which results in a decreased nutritional value in corn and limits its use as a food source. One approach to improving the nutritional value of corn would be to alter, in vitro, the nucleotide sequence of genes that code for the zein proteins to include the codons for lysine and tryptophan. These altered genes would then need to be reintroduced into the corn genome. As a first step to these types of experiments, several clones of zein sequences were constructed in M13mp phage vectors, a pUC plasmid and a pUC-derived expression plasmid. These clones were used to produce zein proteins in E. coil cells. The levels of expression from all three types of the M13mp phage clones were low compared with previous reports using M13mp phage. The lowest level of expression was seen with the recombinant phage M13mp32N/ZG7, which codes for a pre-zein protein with its signal peptide intact. Slightly higher levels of expression were seen with phage coding for fl-galactosidase-zein fusion protein (M13mp10/A20 and M13mp321/A20). Phage isolated from several different M13mp32N/ZG7 plaques were used to infect E. coil cells. Levels of expression from these different isolates, detected by zein antiserum on a Western transfer, ranged from no detectable bands to the presence of two faint bands. These results might have resulted from a promoter mutation in some of the M13mp32N/ZG7 phage molecules. The presence of pre-zein proteins may be harmful to the E. coil cell and therefore result in a selection for reduced levels of expression. There are other possible reasons for the low production of zein in E. coli. One possibility is that zein protein is unstable in E. coll. The higher expression levels seen with the fl-galactosidase-zein protein may be due to alterations in the tertiary structure of this fusion protein which make it less susceptible to proteases. Another possibility is that zein mRNA is unstable. Western transfers of ethanol-soluble protein from cells infected with the pre-zein producing phage M13mp32N/ZG7 show two faint bands (Fig. 7). These two bands may represent processed and unprocessed forms of the ZG7 protein. Protein processing and exporting systems in eucaryotes appear to be similar to those in procaryotes (Inouye and Halegoua, 1980; Fraser and Bruce, 1978; Baty et al., 1981; Talmadge et al., 1980a, 1980b; Roggenkamp et al., 1981). The signal peptides of the zein proteins which have been sequenced so far, including the protein ZG7, show similarities to bacterial sequences. They contain a basic lysine residue near the amino terminus, followed by a sequence of hydrophobic residues and an alanine residue at the cleavage site (Messing et al., 1983). The amino terminus of mature zein proteins, which may also play an important role in export and processing of proteins as proposed by the loop model (Inouye and Halegoua, 1980), is also highly conserved. Therefore, the possibility exists that the ZG7 zein protein was processed in E. coli cells. However, further experiments will be needed to prove that this occurred. These experiments demonstrate for the first time to our knowledge the utility of using the E. coli system for the expression of a protein encoded by a nuclear gene of a higher plant. We now can produce a specific zein polypeptide for use as an antigen. At the present time it is not possible to obtain a single purified zein protein from the mixture that is present in endosperm tissue. The purity of the zein

173 p o l y p e p t i d e p r o d u c e d by E. coli will allow for the p r o d u c t i o n of a n t i b o d i e s d i r e c t e d against a single zein protein. The specificity of such a n t i b o d i e s has yet to be tested. Should they turn out to be specific, then it might be possible to d e t e r m i n e the location of specific zein proteins within the zein p r o t e i n bodies of the e n d o s p e r m tissue. In addition, their coding region can be m a n i p u l a t e d by site-directed m u t a g e n esis to p r o d u c e synthetic zein proteins. W h e n these c o d i n g regions are used as m o l e c u l a r cartridges, they can be moved b e h i n d other p r o m o t e r s which m a y p e r m i t the expression of the zeins in tissue culture rather than d u r i n g e n d o s p e r m d e v e l o p ment. In this case they can be studied directly in p l a n t cells.

Acknowledgements W e thank Kris K o h n a n d Stephanie Y o u n g for help in p r e p a r i n g the m a n u s c r i p t , Beth Lewis for zein antiserum, and D i a n e Vician for help in the D N A synthesis. This work was s u p p o r t e d by the D e p a r t m e n t of Energy D E - F G 0 2 - 8 4 E R 1 3 2 1 0 (J.M.) a n d N I H G M 24756 (I.R.).

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