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The strong LAC4 promoter (PLAC4) from Kluyveromyces lactis has been extensively used to drive expression of heterologous proteins in this industrially important yeast. A drawback of this expression method is the serendipitous ability of PLAC4 to promote gene expression in Escherichia coli. This can interfere with the process of assembling expression constructs in E. coli cells prior to their introduction into yeast cells, especially if the cloned gene encodes a protein that is detrimental to bacteria. In this study, we created a series of PLAC4 variants by targeted mutagenesis of three DNA sequences (PBI, PBII, and PBIII) that resemble the E. coli Pribnow box element of bacterial promoters and that reside immediately upstream of two E. coli transcription initiation sites associated with PLAC4. Mutation of PBI reduced the bacterial expression of a reporter protein (green fluorescent protein [GFP]) by ~87%, whereas mutation of PBII and PBIII had little effect on GFP expression. Deletion of all three sequences completely eliminated GFP expression. Additionally, each promoter variant expressed human serum albumin in K. lactis cells to levels comparable to wild-type PLAC4. We created a novel integrative expression vector (pKLAC1) containing the PLAC4 variant lacking PBI and used it to successfully clone and express the catalytic subunit of bovine enterokinase, a protease that has historically been problematic in E. coli cells. The pKLAC1 vector should aid in the cloning of other potentially toxic genes in E. coli prior to their expression in K. lactis.
For over a decade, the yeast Kluyveromyces lactis has been used for the industrial-scale production of commercially important proteins (14, 16). K. lactis cells rapidly grow to high cell density, efficiently secrete recombinant proteins, and can be easily genetically manipulated, making this organism an attractive eukaryotic host for protein expression. In a typical K. lactis protein expression strategy, a DNA fragment containing the equipment necessary to direct the high-level transcription of a gene of interest is first assembled in Escherichia coli cells prior to its introduction into yeast cells. This fragment typically contains (in 5′ to 3′ order) (i) a strong yeast promoter, (ii) DNA encoding a secretion leader sequence (if secretion of the protein is desired), (iii) the gene encoding the desired protein, (iv) a transcription terminator sequence, and (v) a yeast-selectable marker gene.
In K. lactis, expression of heterologous genes has been achieved using various promoters isolated from native K. lactis genes (7, 10, 11, 15) or using promoters originating from other yeasts (1, 2, 8, 15, 17). However, the K. lactis LAC4 promoter (PLAC4) is often used because of its strength and inducible expression (14). K. lactis PLAC4 drives expression of the LAC4 gene that encodes a native lactase (β-galactosidase [3, 13]) that is an essential part of the lactose-galactose regulon that allows this organism to utilize lactose as a carbon and energy source (4). Two upstream activating sequences (UAS I and UAS II) located in a 2.6-kb intragenic region between LAC4 and LAC12 regulate the transcription of LAC4, which can be induced 100-fold in the presence of lactose or galactose (5).
In addition to its ability to function as a strong promoter in K. lactis, PLAC4 constitutively promotes gene expression in E.coli cells. For example, the K. lactis LAC4 gene was originally isolated by screening a genomic DNA library for clones that were able to functionally complement an E. coli β-galactosidase mutant (3). Bacterial expression of LAC4 was later attributed to nucleotide sequences in PLAC4 that resemble the Pribnow box transcriptional element of bacterial promoters (4).
The ability of PLAC4 to promote gene expression in bacteria can be detrimental to the process of assembling and amplifying yeast expression constructs in E. coli prior to their introduction into yeast cells. This is especially problematic if the cloned gene of interest encodes a translated product that is toxic to E.coli cells. In this study, we addressed this issue by introducing site-directed mutations into PLAC4 to abolish its ability to function in E. coli. We demonstrate that targeted mutagenesis of Pribnow box-like sequences in PLAC4 inhibits the expression of a reporter protein in E. coli but does not affect the promoter's ability to direct the high-level expression and secretion of proteins in K. lactis. Additionally, we demonstrate the use of a PLAC4 mutant to express bovine enterokinase, a commercially important protease that has historically been problematic when expressed in E. coli cells. Finally, we present the construction of an E. coli-K. lactis integrative shuttle vector (pKLAC1) that relies upon a mutant form of PLAC4 to direct protein expression in K. lactis.
The prototrophic K.lactis strain GG799 (MATα [pGKl1+]) is a wild-type industrial isolate (DSM Food Specialties, Delft, The Netherlands) that grows to very high cell density and efficiently secretes heterologous proteins. It was routinely grown and maintained on YPD medium (1% yeast extract, 2% peptone, 2% dextrose) at 30°C. Prior to the transformation of GG799 cells, 1 μg of pGBN1- or pKLAC1-based expression vector containing a gene of interest was linearized by SacII digestion. Linearized expression vectors were used for the integrative transformation of chemically competent K. lactis GG799 cells (New England Biolabs, Ipswich, MA) as directed by the supplier. Colonies of cells transformed with pGBN1, pGBN1PGK1, pGBN1Hyb, pGBN1PBI, or pGBN1PBII-PBIII vectors were selected by growth on YPD agar plates containing 200 μg G418 (Sigma, St. Louis, MO) ml−1 for 2 to 3 days at 30°C. Colonies of cells transformed with pKLAC1-based vectors were selected by growth on agar plates containing 1.17% yeast carbon base (New England Biolabs), 5 mM acetamide, and 30 mM sodium phosphate buffer, pH 7, for 4 to 5 days at 30°C. K. lactis strains expressing heterologous genes were cultured in YP medium containing 2% galactose (YPGal) at 30°C for 48 to 96 h.
Primers used in this study are listed in Table Table1.1. Amplification by PCR was performed using high-fidelity Deep Vent DNA polymerase (New England Biolabs). Typical PCR mixtures contained 0.2 mM deoxynucleoside triphosphates, 0.5 μg of each primer, 1× Thermopol buffer, and 100 ng template DNA in a total reaction volume of 100 μl. Thermocycling typically consisted of a “hot start” at 95°C for 5 min, followed by 30 cycles of successive incubations at 94°C for 30 s, 58°C for 30 s, and 72°C (1 min per kb of DNA). After thermocycling, a final extension was performed at 72°C for 10 min.
All promoter variants were derived from wild-type PLAC4 present in the integrative expression vector pGBN1, a K.lactis-E. coli shuttle vector that contains 2,317 bp of PLAC4 DNA split into 1,663- and 654-bp fragments that are separated by pUC19 plasmid DNA (Fig. (Fig.1).1). Additionally, pGBN1 contains DNA encoding the Saccharomyces cerevisiae α-mating factor (α-MF) pre-pro domain immediately downstream of PLAC4 to direct the secretion of heterologously expressed proteins. Finally, pGBN1 carries a Geneticin (G418) resistance gene expressed from the S. cerevisiae ADH2 promoter for dominant selection in yeast.
To create plasmid pGBN1PGK1, a PmlI-HindIII fragment containing 488 base pairs of the S. cerevisiae PGK1 promoter was cloned into the HpaI-HindIII sites of plasmid pGBN1 to replace 1,067 base pairs of native PLAC4 (Fig. (Fig.2B).2B). Primer P1 and primer P2 were used to amplify 283 base pairs of the S. cerevisiae PGK1 promoter using plasmid pGBN1PGK1 as template. The 283-bp fragment was cloned into the SnaBI-HindIII sites of plasmid pGBN1 to produce plasmid pGBN1Hyb. Primer P3 was designed to incorporate mutations into the putative Pribnow box-like sequence (PBI) that lies upstream of the E. coli major transcription start site as detailed in Fig. Fig.2B.2B. Primers P2 and P3 were used to amplify a PLAC4 fragment containing mutations in PBI using plasmid pGBN1 as template. Amplified DNA from this initial PCR was used as template for a second PCR using primers P2 and P4. The final DNA product was cloned into the SnaBI-HindIII sites of plasmid pGBN1 to produce plasmid pGBN1PBI. A PCR-knitting method was used to mutate the PBII and PBIII sequences (Fig. (Fig.2B)2B) that lie upstream of the E. coli minor transcription start site using complementary primers P5 and P6. Primers P2 and P5 and primers P4 and P6 were used to amplify 586-bp and 160-bp mutated PLAC4 DNA fragments, respectively. Each amplified DNA product was purified by QIAquick PCR purification spin column chromatography (QIAGEN, Valencia, CA) and combined as template in a second amplification reaction containing primers P2 and P4. The amplified PLAC4 DNA fragment containing mutagenized PBII and PBIII sites was cloned into the SnaBI-HindIII sites of plasmid pGBN1 to produce plasmid pGBN1PBII-PBIII.
Green fluorescent protein (GFP) was PCR amplified with primers P7 and P8 using plasmid pGFPuv (Clontech, Palo Alto, CA) as template. Amplified GFP was cloned in frame with the α-MF pre-pro domain in the BglII-NotI sites of the various pGBN vectors (see previous section). Lysates of bacteria containing various pGBN-GFP constructs were prepared from 50-ml overnight cultures grown at 30°C in Luria-Bertani medium containing 100 μg ml−1 ampicillin. Cultures were centrifuged, and the cell pellets were frozen on dry ice, thawed at room temperature, and resuspended in 10 ml of lysis buffer (20 mM Tris-HCl, pH 7.5, containing 50 mM NaCl and 1 mM EDTA). The cells were disrupted with a Sonicator (Heat Systems-Ultrasonics, Plainview, NY) for 15 s on setting 7, and cell debris was removed by centrifugation at 15,000 × g for 10 min. The protein concentration of each lysate was determined by measuring its absorbance at 280 nm. Proteins (100 μg) in each lysate were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10 to 20% polyacrylamide gradient gel, transferred to nitrocellulose, and blocked overnight in phosphate-buffered saline containing 0.05% Tween 20 (PBS-T) and 5% nonfat milk (wt/vol) at 4°C. An anti-GFP monoclonal antibody (Clontech) diluted 1:1,000 in PBS-T containing 5% nonfat milk was used to probe the blot, followed by incubation with a horseradish peroxidase-coupled anti-mouse secondary antibody (KPL, Gaithersburg, MD) diluted 1:2,000 in PBS-T containing 5% nonfat milk. Protein-antibody complexes were detected using LumiGlo detection reagents (Cell Signaling Technology, Beverly, MA). The amount of GFP produced in E. coli was measured by densitometry using Molecular Imager FX (Bio-Rad, Hercules, CA) and Quantity One software.
Primers P9 and P10 were used to amplify the gene encoding human serum albumin (HSA) that was subsequently cloned in frame with the α-MF sequence in the XhoI-NotI sites of the various pGBN vectors. Primer P9 was designed to encode the K. lactis Kex1 protease cleavage site (KR↓) immediately upstream of the HSA open reading frame to ensure correct processing of the protein in the Golgi apparatus. K. lactis strains containing integrated pGBN-HSA DNA were grown in 2-ml cultures of YPGal for 48 h at 30°C. The level of HSA secretion was visually assessed by separation of proteins in 15 μl of spent culture medium by SDS-PAGE on a 10 to 20% polyacrylamide gradient gel followed by Coomassie blue staining.
A DNA fragment encoding the enterokinase catalytic subunit (EKL) was PCR amplified with primers P11 and P12 and cloned in frame with the α-MF pre-pro domain in the XhoI-BglII restriction sites of the various pGBN vectors containing the PLAC4 variants or in the vector pKLAC1 (see below). The DNA sequence of EKL in the various pGBN-EKL or pKLAC1-EKL vectors was confirmed by nucleotide sequencing. Secretion of enterokinase by K. lactis strains containing integrated pKLAC1-EKL constructs was assessed by growing cells in 2 ml YPGal for 48 h at 30°C and assaying spent culture medium for enterokinase activity as described below.
Spent culture medium was isolated by microcentrifugation of 1 ml of a saturated culture of pKLAC1-EKL-integrated K. lactis at 15,800 × g for 1 min to remove cells. Enterokinase activity was measured using the fluorogenic peptide substrate GDDDDK-β-napthylamide (Bachem, King of Prussia, PA). Spent culture medium (50 μl) was mixed with 50 μl enterokinase assay buffer (124 mM Tris-HCl, pH 8.0, containing 0.88 mM GD4K-β-napthylamide and 17.6% dimethyl sulfoxide), and fluorescence intensity (excitation, 337nm; emission, 420 nm) was measured over time. A comparison of the amount of enzyme activity associated with measured quantities of purified enterokinase (New England Biolabs) to the activity present in spent K. lactis culture medium was used to estimate the amount of active enterokinase secreted by K. lactis strains. To compensate for a mild inhibitory effect that YPGal culture medium has on the enterokinase assay, purified enterokinase was first diluted into spent medium from a culture of untransfected K. lactis cells prior to measuring enterokinase activity as described above.
Vector pKLAC1 was created by replacing the S. cerevisiae α-MF pre-pro domain and the G418 resistance gene of vector pGBN1PBI with the K. lactis α-MF pre-pro domain and the Aspergillus nidulans acetamidase gene (amdS), respectively. DNA encoding the K. lactis α-MF pre-pro domain was PCR amplified from K. lactis genomic DNA using primers 13 and 14 and cloned into the SacI-XhoI sites of pLitmus29 (New England Biolabs). The cloned K. lactis α-MF sequence was subsequently excised by HindIII and XhoI digestion and cloned into the HindIII-XhoI sites of plasmid pGBN1PBI to produce plasmid pGBN1PBI-KlαMF. A 1,520-bp DNA fragment containing all of the A. nidulans amdS gene except the first 128 bp was amplified using primers P15 and P16 and a cloned amdS gene as template (kindly provided by Peter Dekker of DSM Food Specialties, Delft, The Netherlands). This fragment was cloned into the BamH I-SmaI sites of plasmid pGBN1PBI-KlαMF, replacing the G418 resistance gene and producing plasmid pGBN1PBI-KlαMF-1520. The remaining 128 bp of the 5′ end of the amdS gene was amplified by PCR with primers P16 and P17, digested with BamHI, and cloned into the BamHI site of vector pGBN1PBI-KlαMF-1520, and the proper orientation of the fragment was confirmed by DNA sequencing.
The resulting vector was named pKLAC1 (GenBank accession no. AY968582).
The initiation sites of two RNA transcripts (a major and a minor transcript) associated with the E. coli expression of K.lactis PLAC4 have been previously mapped (4). Three DNA sequences (PBI, PBII, and PBIII) that resemble bacterial Pribnow box transcriptional elements lie approximately 10 bp upstream of the major (PBI) and minor (PBII and PBII) transcription initiation sites (Fig. (Fig.2A).2A). We therefore generated a series of four PLAC4 variants aimed at eliminating the E. coli promoter activity of PLAC4 by either replacing or introducing point mutations in PBI and PBII/PBIII as shown in Fig. Fig.2B.2B. Vector pGBN1PGK1 incorporates 485 bp of the S. cerevisiae PGK1 promoter (PPGK1) in place of 1,067 bp of native PLAC4, thereby removing both galactose-responsive upstream activating sequences (UAS I and UAS II) and all three Pribnow box-like sequences. Vector pGBN1Hyb incorporates 283 bp from the 5′ end of PPGK1 in place of 283 bp comprising the 5′ end of PLAC4, resulting in the deletion of all three Pribnow box-like sequences but leaving both UAS intact. Vector pGBN1PBI contains 6 point mutations that eliminate the Pribnow consensus sequence of PBI between nucleotides −201 and −210 of PLAC4. Similarly, vector pGBN1PBII-PBIII contains 5 point mutations that eliminate the Pribnow consensus sequences of PBII and PBIII between nucleotides −139 and −144 of PLAC4.
Each PLAC4 variant was tested for its ability to drive the E.coli expression of a reporter gene encoding GFP that was cloned in frame with the S. cerevisiae α-mating factor pre-pro domain in each of the pGBN vectors. The presence of GFP produced from PLAC4 variants in E. coli lysates was analyzed by Western blot analysis. Removal of the PBI sequence by mutation resulted in an 87% decrease in GFP expression (Fig. (Fig.3A,3A, lane 5), as determined by densitometry, relative to GFP produced by the wild-type PLAC4 (Fig. (Fig.3A,3A, lane 2). However, mutation of both PBII and PBIII sequences (Fig. (Fig.3A,3A, lane 6) did not detectably down-regulate GFP expression. Deletion of all three Pribnow box-like sequences from PLAC4 by replacement with PPGK1 DNA (Fig. (Fig.3A,3A, lanes 3 and 4) led to a complete loss of detectable GFP expression. These results indicate that the majority of PLAC4 expression in E. coli is dependent upon the presence of the PBI sequence.
To test whether the PLAC4 variants were able to direct the expression of a heterologous gene in K. lactis, the gene encoding HSA was cloned into each of the pGBN vectors. HSA was chosen as a reporter protein due to its high-level expression and efficient secretion from K. lactis when expressed from wild-type PLAC4 (7). K. lactis strains containing each of the integrated pGBN1-HSA expression vectors were grown to saturation in YPGal medium, and secreted proteins in the spent culture medium were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and detected by Coomassie blue staining. HSA migrates as a 66-kDa band that can be readily detected in unconcentrated spent culture medium, and its identity was confirmed by Western blotting with an anti-HSA antibody (data not shown). K. lactis strains containing integrated pGBN1PBI-HSA, pGBN1Hyb-HSA, and pGBN1PBII-PBIII-HSA vectors secreted HSA in amounts comparable to a control strain harboring pGBN1-HSA where HSA is expressed from wild-type PLAC4 (Fig. (Fig.3B,3B, lane 2). These data indicate that mutation or deletion of the PBI, PBII, and PBIII sequences of PLAC4 does not significantly alter the promoter's ability to function in K. lactis. It is also noteworthy that markedly less HSA was secreted from cells harboring pGBN1PGK1-HSA (Fig. (Fig.3B,3B, lane 3) compared to cells expressing HSA from either wild-type PLAC4 (Fig. (Fig.3B,3B, lane 2) or the other PLAC4 variants (Fig. (Fig.3B,3B, lanes 4 through 6). This is consistent with the notion that HSA expression from PPGK1 is suppressed in galactose-containing medium because both UAS required for galactose-induced expression have been deleted.
Bovine enterokinase is an important protease that is often used to cleave affinity tags from engineered fusion proteins. Production of enterokinase in E. coli is plagued by low yields that are attributable to the protein's toxicity in bacteria. Therefore, we sought to use the expression of enterokinase in K. lactis as a means to circumvent its poor expression in bacteria. Numerous attempts to assemble K. lactis expression vectors in E. coli, where DNA encoding the enterokinase light chain (EKL) was placed downstream of wild-type PLAC4, resulted in widespread isolation of clones containing loss-of-function mutations (e.g., frame shifts or early terminations) within the EKL coding sequence. We reasoned that the ability of PLAC4 to promote gene expression in E. coli was likely creating selective pressure against the propagation of intact EKL-containing clones. We therefore examined whether the PLAC4 variants that exhibited reduced or abolished expression in E.coli could be used to facilitate cloning of the toxic EKL gene into K. lactis expression vectors in E. coli prior to their introduction into yeast.
The EKL gene was PCR amplified using a high-fidelity polymerase and cloned downstream of the various PLAC4 variants in the pGBN1 vectors (Fig. (Fig.2B).2B). The entire EKL gene (708 bp) of numerous isolated clones was sequenced to determine the presence of loss-of-function mutations. When cloned under the control of wild-type PLAC4 in pGBN1, 11 of 12 (92%) clones examined contained loss-of-function mutations. However, no mutations were found in EKL cloned in vectors pGBN1PGK1 (nine clones sequenced) or pGBN1Hyb (seven clones sequenced), vectors containing PLAC4 variants that completely lack E. coli promoter function. Additionally, no mutations were found in EKL cloned in vector pGBN1PBI (nine clones sequenced) where E. coli expression is reduced ~87% due to mutations in PBI. Additionally, 3 of 10 (30%) EKL clones in pGBN1PBII-PBIII contained loss-of-function mutations. Together, these data show that the function of wild-type PLAC4 in E. coli adversely affects the cloning efficiency of a toxic gene and indicate that PLAC4 variants that either lack or have severely reduced function in E. coli are better suited for the assembly of K. lactis expression constructs in bacteria.
Relying upon the findings of this study, we assembled a novel K. lactis integrative expression vector (pKLAC1) for the secretion of proteins from K. lactis (Fig. (Fig.4).4). This vector is based on the PLAC4-PBI variant that contains mutations in PBI (Fig. (Fig.2B,2B, pGBN1PBI) and contains (in 5′-to-3′ order) a PBI-deficient LAC4 promoter, the K. lactis α-mating factor secretion leader sequence, a multiple cloning site, the K. lactis LAC4 transcription terminator, a selectable marker cassette containing the Aspergillus nidulans acetamidase gene (amdS) expressed from the S. cerevisiae ADH2 promoter (PADH2), and an E. coli origin of replication and ampicillin resistance gene to allow for its propagation in E. coli. Digestion of this vector with SacII or BstX I generates a linear expression cassette that integrates into the promoter region of the LAC4 locus of the K.lactis chromosome upon its introduction into K. lactis cells. Transformed yeast cells are isolated by nitrogen source selection on yeast carbon base medium containing 5 mM acetamide, which can be converted to a simple nitrogen source only if the expression cassette (containing the amdS gene) has integrated into the chromosome (12).
Vector pKLAC1 was used to secrete enterokinase from K.lactis cells after successfully assembling the expression vector in E. coli (pKLAC1-EKL). Strains harboring integrated pKLAC1-EKL were cultured in YPGal medium for 2 days. Enterokinase proteolytic activity in the spent culture medium was assayed by measuring the rate of cleavage of a fluorogenic peptide. Measurements of activity performed on culture supernatant from seven pKLAC1-EKL integrated strains showed that all seven secreted active enterokinase (Fig. (Fig.5).5). However, two of the seven strains (KlEK-S1 and KLEK-S4) secreted greater levels of enterokinase activity than the other five. Southern analysis determined that strains KLEK-S1 and KLEK-S4 contained multiple tandem copies of integrated pKLAC1-EKL (data not shown). The yield of enterokinase secreted from strain KLEK-S1 grown in shake flasks was estimated to be ~1.1 mg/liter based on a comparison of secreted enzyme activity to the activity of known quantities of purified enterokinase as described in Materials and Methods.
An important component of any yeast-based expression system is a shuttle vector that allows for the propagation of cloned genes in bacteria prior to their introduction into yeast cells for expression. In this context, yeast expression systems that utilize the strong K. lactis LAC4 promoter (PLAC4) can be adversely affected by the serendipitous function of PLAC4 in E. coli. This promoter activity can interfere with the cloning efficiency of genes whose translational products are potentially detrimental to bacteria. In the present study, we addressed this by eliminating sequences within PLAC4 that resemble the bacterial Pribnow box transcription element. We showed that PLAC4 variants that lack Pribnow box-like sequences have reduced or abolished ability to promote gene expression in E. coli but retain their full ability to function as strong promoters in yeast. Finally, we constructed a K. lactis expression vector that is based upon the PLAC4-PBI variant created in this study and used it to express the commercially important protease enterokinase.
The Pribnow box is an important component of bacterial promoters, that is, an A/T-rich region located approximately 10 nucleotides upstream from the site where transcription begins. A prior study mapped a major and a minor transcription start site associated with the K. lactis LAC4 promoter in E. coli (4). Two stretches of nucleotide sequence that closely resemble the Pribnow box consensus sequence TATAAT are located at −204 to −209 (PBI) and −136 to −144 (PBII and PBIII) within PLAC4 and reside just upstream of the major and minor transcription start sites, respectively. Elimination of these Pribnow box-like sequences by targeted mutagenesis revealed that most PLAC4-based expression in E. coli was due to the PBI sequence associated with the major transcription start site. Interestingly, mutation of PBII and PBIII did not lead to a significant decrease in the expression of GFP in E. coli but nevertheless led to a 62% decrease in the isolation of clones carrying loss-of-function mutations in the EKL gene. This suggests that the cloning efficiency of detrimental genes in E. coli can be dramatically improved even by small decreases in PLAC4 expression levels. Importantly, none of the point mutations that reduced or eliminated PLAC4 expression in E. coli adversely affected the levels of protein expression and secretion from K. lactis cells. This finding underscores differences in promoter elements that are required for bacterial versus yeast expression. For example, in K. lactis, PLAC4 initiates transcription at multiple sites (−97, −98, −105, −115, and −127) presumably due to TATA box sequences in the −169 to −173 and −226 to −234 regions (6) that are distinct from the PBI and PBII/PBIII sequences that are located within the −204 to −209 and −136 to −144 regions, respectively.
A recent study used a different method to curtail the potentially detrimental effects of PLAC4 expression in E. coli (9). In this work, a yeast intron containing translational stop codons was placed immediately downstream of the translational start codon of the desired protein. Because E. coli cells cannot process introns, PLAC4 activity generated an mRNA containing early stop codons that prevented translation of the full-length protein in bacteria. This method was effective in lowering the PLAC4-based expression of xylanase and lipase genes in E. coli; however, xylanase activity was not completely abolished. The authors noted that this was likely due to alternative translational start codons that lie downstream of the inserted intron that may allow for translation of active protein fragments (9). In contrast, the promoter variants described in the present study presumably function by blocking the ability of PLAC4 to initiate transcription in E. coli, which provides a tighter regulation of the expression of potentially detrimental recombinant proteins.
Based on our findings, we constructed a novel K. lactis integrative expression vector (pKLAC1) for the production of recombinant proteins. Two key elements of this vector are as follows: (i) the PLAC4-PBI variant containing mutations in PBI to allow the assembly of DNA fragments encoding potentially toxic proteins in E. coli and high-level protein production in yeast and (ii) an acetamidase-selectable marker gene. Expression of acetamidase in transformed yeast cells allows for their growth on medium lacking a simple nitrogen source but containing acetamide (12). Acetamidase breaks down acetamide to ammonia, which can be utilized by cells as a source of nitrogen. An important benefit of this selection method is that it enriches transformant populations for cells that have incorporated multiple tandem integrations of a pKLAC1-based expression vector and that produce more recombinant protein than single integrations (Fig. (Fig.55 and data not shown). We have recently shown that more than 90% of transformants that form on acetamide plates following transformation of K. lactis strain GG799 with pKLAC1-based constructs that express HSA or the E. coli maltose binding protein contain two to four copies of the integrated vector.
We successfully used pKLAC1 to efficiently clone the toxic protease enterokinase in E. coli and secrete it from K. lactis cells. Additionally, the use of pKLAC1 is applicable to the expression of other recombinant proteins that are problematic in E. coli due to their toxicity or other detrimental effects on bacterial cells. For example, we have recently utilized pKLAC1 to successfully clone and express in K.lactis the gene encoding mouse transthyretin following numerous unsuccessful attempts using various prokaryote-based systems (J. Ingram, P. A. Colussi, C. H. Taron, and B. Slatko, unpublished data). Additionally, pKLAC1 has been used to clone and express in K.lactis toxic glue proteins from marine organisms (J. Platko, personal communication) and a multifunctional bacterial cellulase (D. Distel, personal communication) that were unable to be expressed using various prokaryotic expression systems.
C.H.T. is grateful to Donald Comb for support. We thank B. Taron, A. Fabre, L. McReynolds, B. Slatko, F. Stewart, and D. Distel for comments on the manuscript.