Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.9 (enterokinase)
 675 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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 approximately 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 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.
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PMID:Kluyveromyces lactis LAC4 promoter variants that lack function in bacteria but retain full function in K. lactis. 1626 45

hLM (human lysozyme) has important potential application as a future safely administered human drug and food additive. To produce secreted rhLM (recombinant hLM) from the yeast Pichia pastoris, the signal peptide from HSA (human serum albumin) was employed to direct secreted expression. On the basis of the vector pPIC3.5k, an overexpression vector, pPIC3.5k-hLM, carrying the strong promoter AOX1 (aldehyde oxidase 1), the HSA signal peptide, the enterokinase recognition motif, the lysozyme gene and other necessary genetic segments was constructed and this was followed by a series of genetic manipulations. A positive colony was picked off to test its expression pattern. The target protein, rhLM, was obtained from the supernatant and showed a gradual enrichment with the induction time course, reaching its highest level at 72 h. This pattern was identical with that shown by the secreted expression of a heterologous protein directed by Saccharomcyes cerevisiae a-mating factor prepro-signal peptide in P. pastoris. After a series of purification processes, including ultrafiltration with a hollow-fibre membrane module, DEAE-Sepharose, Sephadex G50 chromatography and enterokinase digestion, the mature protein was characterized by MALDI-TOF-MS/MS (matrix-assisted laser-desorption ionization-time-of-flight tandem MS), N-terminal amino acid sequencing, and K(m) and K(cat) determination. The results confirmed that the rhLM was identical with native hLM. Moreover, the mature protein exhibited in vitro bacteriolytic activity against the Gram-positive bacterium Micrococcus lysodeikticus and the Gram-negative bacterium Escherichia coli. Taken together, it appeared that the HSA signal peptide was able direct secretive expression of a heterologous protein in P. pastoris.
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PMID:Secreted expression of human lysozyme in the yeast Pichia pastoris under the direction of the signal peptide from human serum albumin. 1824 31