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Integrative Candida maltose gene expression system and applications thereof

A technology of Candida and maltose, applied in the biological field

Active Publication Date: 2013-04-17
FUJIAN FUDA BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, there is no report on the expression of heterologous proteins in integrated Candida maltosa so far in the art

Method used

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  • Integrative Candida maltose gene expression system and applications thereof
  • Integrative Candida maltose gene expression system and applications thereof
  • Integrative Candida maltose gene expression system and applications thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] Embodiment 1, preparation Candida maltosa auxotrophic strain

[0112] Candida maltosa cells (CGMCC No.2.1371, China General Microbiology Collection Center) were cultured in YPD medium (1% (w / v) yeast powder, 2% (w / v) peptone, 2% (w / v) glucose ) at 28°C for 16 hours. Cells were collected by centrifugation and resuspended in sterile water. After measuring the cell concentration by counting, they were spread and inoculated on a YPD medium plate. Place the plate under a UV lamp (using a G8T5 UV germicidal lamp (Sylvania) and an irradiation distance of 30 cm) for different time periods of irradiation. After irradiation, the plate was cultured at 28°C, the number of clones was measured, and the survival rate was calculated based on this.

[0113] After the cells were diluted, the YEPD medium plate was spread and inoculated, and MM (1.34% (w / v) yeast nitrogen source without histidine, 2% (w / v) glucose, 0.002% ( w / v) biotin) medium plate, subculture the auxotrophic strain. ...

Embodiment 2

[0114] Embodiment 2, the cloning of Candida maltosa His5 gene

[0115] Candida maltosa (auxotrophic type) was cultured overnight, the cells were collected, and the genomic DNA was isolated and extracted, and then digested with the restriction endonuclease EcoRI. A genomic library of Candida maltosa was constructed in λ phage (Lamda GT 11, obtained from Invitrogen). The artificially synthesized His5 oligonucleotide of Candida maltosa IAM 12247 (T.Hikiji et al.1989, Curr Genet 16:261-266) has the sequence: 5'-AAAACTGGTATCTTGTTAGATGCTAATGAAAATACTCATGGTCC-3'(NCBI: X17310. 1) (SEQ ID NO: 10), the His5 gene fragment of Candida maltosa CGMCC No.2.1371 was screened from the genome library. PCR primer 5'-A was designed according to the sequence of the His5 gene fragment of Candida maltosa in the λ construction library GCTAGC TTGACTCATACTTGAGCGCGGTAAAG-3' (SEQ ID NO: 11) and 5'-A GAATTCTTC ATCATGAACAAGGAAATCTTTTCTTAG-3' (SEQ ID NO: 12) (respectively containing enzyme cutting sites ...

Embodiment 3

[0116] Embodiment 3, synthetic GAL1 promoter

[0117] From the Candida maltosa genomic library described above, a GAL1 (encoding galactokinase) gene fragment was screened. Synthesize GAL1 oligonucleotide according to the GAL1 gene sequence of Candida maltosa IAM 12247, its sequence is: 5'-TTTTATTCCAACGTCGAAACCTAATCAACAGAGATTCACTGAAGTC-3' (SEQ ID NO: 13) (NCBI: D29759.1), mark with P32 (first use Phosphomonoesterase PMase removes the free phosphate radical of the terminal nucleotide of the oligonucleotide fragment, exposing -OH, and then uses T4 polynucleotide kinase and ATP labeled with radioactive isotope P32 (abbreviated as γ-P32) to convert the oligonucleotide to The 5' position of the 5' terminal nucleotide of the nucleotide fragment is connected with γ-P32 to obtain a P32-labeled oligonucleotide fragment), which is used for the screening of the GAL1 gene fragment.

[0118] The GAL1 gene fragment was cloned into the pUC19 vector, and the DNA sequence was determined. Acco...

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Abstract

The invention relates to an integrative Candida maltose gene expression system and applications thereof. The system contains Candida maltose of histidine auxotroph and an expression construct, wherein the expression construct contains the following elements: Origin, a GAL1 promoter, an alpha-binding factor secretion guide sequence, terminator and a Candida maltose His5 gene. Verifications prove that the expression system can be used in the expression of various proteins and the expression system has high expression efficiency and expression level.

Description

technical field [0001] The invention belongs to the field of biotechnology; more specifically, the invention relates to the construction of a new integrated Candida maltosa gene expression system and its application in protein production. Background technique [0002] Since the first description of the non-spore-forming yeast Candida maltosa by Komagata et al. more than 40 years ago (1964), it has aroused extensive academic and industrial interest. Candida maltosa can grow under the condition of n-alkane or fatty acid as the sole carbon source and energy. The first Candida maltosa isolate was obtained from the glue in the neutralization tank of the monosodium glutamate manufacturing process. In the early 1960s, hydrocarbon biochemistry became the subject of industrial research, with most oil companies working on the potential application of microorganisms for the production of single-cell proteins for food (animal feed) and for the use of amino acids, fatty acids, sterols a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/81C12R1/72
Inventor 叶秀云靳伟刚陈萍张洋罗鋆琳应喜娟傅仙玉
Owner FUJIAN FUDA BIOTECH