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Method for construction and use of kluyveromyces lactis promoter variants in k. lactis that substantially lack e. coli transcriptional capability

A Kluyveromyces, promoter technology, applied in chemical instruments and methods, botanical equipment and methods, biochemical equipment and methods, etc., can solve problems such as destroying the functional expression of potentially toxic recombinant proteins

Inactive Publication Date: 2007-04-25
NEW ENGLAND BIOLABS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Unfortunately, this modification disrupts the functional expression of some but not all potentially toxic recombinant proteins

Method used

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  • Method for construction and use of kluyveromyces lactis promoter variants in k. lactis that substantially lack e. coli transcriptional capability
  • Method for construction and use of kluyveromyces lactis promoter variants in k. lactis that substantially lack e. coli transcriptional capability
  • Method for construction and use of kluyveromyces lactis promoter variants in k. lactis that substantially lack e. coli transcriptional capability

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Experimental program
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Embodiment

[0040] Yeast strains, transformation and culture conditions

[0041] [39] Prototrophic K. lactis strain GG799 (MATα[pGK11+]) was grown and maintained in YPD medium (1% yeast extract, 2% peptone, 2% glucose) in a conventional manner at 30°C . Before transforming GG799 cells, 1 μg of pGBN1- or pKLAC1-based expression vector containing the gene of interest was linearized by SacII digestion. The linearized expression vector was used to transform commercially obtained competent K. lactis GG799 cells (New England Biolabs, Beverly, MA) in an integrative fashion according to the supplier's instructions. Passed at 30°C in 18ml containing 200μg G4 -1 (Sigma, St.Louis, MO) YPD agar plate cultured for 2-3 days, selected with pGBN1, pGBN1 PGK1 , pGBN1 杂合 , pGBN1 PBI or pGBN1 PBII-PBIII Vector-transformed cell colonies. By culturing at 30° C. on agar plates containing 1.17% yeast carbon base (New England Biolabs, Beverly, MA), 5 mM acetamide (New England Biolabs, Beverly, MA) and 30 ...

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Abstract

Methods and compositions are provided relating to production of recombinant protein in yeast. A modified PLAC4 is described where one or more mutations may be introduced into the Pribnow box-like sequences in the promoter. The modified promoter when placed upstream of a target gene in a vector causes a significant reduction of target gene expression in transformed bacteria but produces efficient expression of the target gene in yeast.

Description

Background of the invention [0001] [1] For decades, the budding yeast Kluyveromyces lactis (K. lactis) has been widely used in the food and dairy industries for the production of recombinant proteins on an industrial scale for reasons including the following factors : (i) many strains of K. lactis grow rapidly in culture and grow to extremely high cell densities; (ii) K. lactis efficiently directs protein secretion into the medium; and (iii) lactic acid Kluyveromyces has GRAS (Generally Recognized as Safe ( G generally R egarded A the s S afe)) FDA qualification, which allows it to be used in food, agricultural and health-related applications. [0002] [2] A typical K. lactis heterologous protein production strategy involves secretion of the desired protein from the cells into the growth medium. This method has a number of advantages over intracellular expression methods: (i) the protein produced is fairly pure, since K. lactis secretes relatively small amounts of endogen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H21/04C07H15/10C12N15/63C12N1/21C12N1/15C12N1/19C12N15/00C07K14/39C12N1/18C12N15/74C12N15/81C12P21/02C12P21/06
CPCC12N15/81C12P21/02C12N2820/702C12N2800/102C12N2800/101C07K14/39
Inventor C·H·塔龙P·A·科卢斯
Owner NEW ENGLAND BIOLABS
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