High-level secretion of correctly processed recombinant human interleukin-1β in Kluyveromyces lactis

The strong LAC4 promoter (P LAC4 ) 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 P LAC4 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 P LAC4 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 P LAC4 . Mutation of PBI reduced the bacterial expression of a reporter protein (green fluorescent protein [GFP]) by ϳ87%, 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 P LAC4 . We created a novel integrative expression vector (pKLAC1) containing the P LAC4 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.

mentioning

confidence: 99%

Kluyveromyces lactis LAC4 Promoter Variants That Lack Function in Bacteria but Retain Full Function in K. lactis

Colussi

Taron

2005

“…We therefore analyzed the secretory capabilities of the dgr151-1 strain by utilizing two other reporter proteins, IL-1␤ and HSA. Wild-type and dgr151-1 cells were transformed with the plasmid pYG81, which carries the IL-1␤ cDNA fused to the secretion signal of the K. lactis killer toxin (␣ subunit) (19). The Table 1]) were transformed with the plasmid pGM-GAM carrying the GAA gene of A. adeninivorans.…”

Section: Resultsmentioning

confidence: 99%

“…The yeast K. lactis is one of the model systems utilized for heterologous protein production in the food and pharmacology industries (18,19,35,39,44,51). In this work, we reported the characterization of a non-glucose-repressible mutant of K. lactis to be used as a host for recombinant protein production.…”

Section: Discussionmentioning

confidence: 99%

“…Plasmids and transformations. The plasmids used for this study were pYG107, carrying a cassette for the secreted expression of recombinant human serum albumin (HSA) driven by the native prepro signal sequence under the control of the LAC4 promoter, as described by Fleer et al (18), and pYG81, carrying the secretion cassette of interleukin-1␤ (IL-1␤) with the K. lactis killer toxin signal sequence under the control of the PHO5 promoter (19). The K. lactis-E. coli shuttle vector pGM-GAM contained the glucoamylase (GAA) gene from the yeast Arxula adeninivorans (33).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Improved Production of Heterologous Proteins by a Glucose Repression-Defective Mutant of Kluyveromyces lactis

Donnini

Farina

Neglia

et al. 2004

The secreted production of heterologous proteins in Kluyveromyces lactis was studied. A glucoamylase (GAA) from the yeast Arxula adeninivorans was used as a reporter protein for the study of the secretion efficiencies of several wild-type and mutant strains of K. lactis. The expression of the reporter protein was placed under the control of the strong promoter of the glyceraldehyde-3-phosphate dehydrogenase of Saccharomyces cerevisiae. Among the laboratory strains tested, strain JA6 was the best producer of GAA. Since this strain is known to be highly sensitive to glucose repression and since this is an undesired trait for biomass-oriented applications, we examined heterologous protein production by using glucose repression-defective mutants isolated from this strain. One of them, a mutant carrying a dgr151-1 mutation, showed a significantly improved capability of producing heterologous proteins such as GAA, human serum albumin, and human interleukin-1␤ compared to the parent strain. dgr151-1 is an allele of RAG5, the gene encoding the only hexokinase present in K. lactis (a homologue of S. cerevisiae HXK2). The mutation in this strain was mapped to nucleotide position ؉527, resulting in a change from glycine to aspartic acid within the highly conserved kinase domain. Cells carrying the dgr151-1 allele also showed a reduction in N-and O-glycosylation. Therefore, the dgr151 strain may be a promising host for the production of heterologous proteins, especially when the hyperglycosylation of recombinant proteins must be avoided.Yeasts are very useful hosts for the production of heterologous proteins. The yeast Kluyveromyces lactis presents several advantages over other yeast species. It is positive for lactose fermentation, is able to grow on cheap substrates such as residual whey from dairy industries, and has competitive secretory properties, excellent large-scale fermentation characteristics, and food grade status; also, both episomal and integrative expression vectors are available for it (for reviews, see references 20, 40, and 50). Its ability to secrete heterologous proteins into the medium at a concentration higher than that secreted by Saccharomyces cerevisiae was demonstrated previously (50), although the secretory and glycosylation processes and their regulation are still poorly understood for K. lactis (1,42,43).For K. lactis, the regulation of primary carbon metabolism differs markedly from that for S. cerevisiae and reflects the dominance of respiration over fermentation that is typical for the majority of yeast species (7). In K. lactis, respiration is not repressed by glucose, and fermentative and oxidative metabolism can take place simultaneously. Glucose repression, however, does exist: several enzymes that are required for alternate carbohydrate metabolism have been shown to be subject to glucose repression (6,13,17,25,30). The K. lactis genes involved in glucose repression include RAG1, encoding a lowaffinity glucose permease (23, 48); DGR151 (or RAG5), encoding the single hexokinase of this yea...

“…Kluyveromyces lactis is an industrially important yeast due to its ability to grow to a very high cell density and abundantly secrete heterologous proteins (10,25,30,31). We are interested in using K. lactis to secrete commercially important recombinant proteins carrying ChBD affinity tags to facilitate their capture on chitin beads.…”

mentioning

confidence: 99%

Characterization of a Nucleus-Encoded Chitinase from the Yeast Kluyveromyces lactis

Colussi

Specht

Taron

2005

Endogenous proteins secreted from Kluyveromyces lactis were screened for their ability to bind to or to hydrolyze chitin. This analysis resulted in identification of a nucleus-encoded extracellular chitinase (KlCts1p) with a chitinolytic activity distinct from that of the plasmid-encoded killer toxin ␣-subunit. Sequence analysis of cloned KlCTS1 indicated that it encodes a 551-amino-acid chitinase having a secretion signal peptide, an amino-terminal family 18 chitinase catalytic domain, a serine-threonine-rich domain, and a carboxy-terminal type 2 chitin-binding domain. The association of purified KlCts1p with chitin is stable in the presence of high salt concentrations and pH 3 to 10 buffers; however, complete dissociation and release of fully active KlCts1p occur in 20 mM NaOH. Similarly, secreted human serum albumin harboring a carboxy-terminal fusion with the chitin-binding domain derived from KlCts1p also dissociates from chitin in 20 mM NaOH, demonstrating the domain's potential utility as an affinity tag for reversible chitin immobilization or purification of alkaliphilic or alkali-tolerant recombinant fusion proteins. Finally, haploid K. lactis cells harboring a cts1 null mutation are viable but exhibit a cell separation defect, suggesting that KlCts1p is required for normal cytokinesis, probably by facilitating the degradation of septum-localized chitin.