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Novel prolipase-bovine trypsinogen fusion proteins

A kind of technology of serine protease and lipase, applied in the field of fusion protein

Active Publication Date: 2012-05-30
BIOCON LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The current state of the art fails to provide an acceptably satisfactory / desirable production method capable of expressing a purifiable form of the heterologous fusion polypeptide in a suitable host cell

Method used

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  • Novel prolipase-bovine trypsinogen fusion proteins
  • Novel prolipase-bovine trypsinogen fusion proteins
  • Novel prolipase-bovine trypsinogen fusion proteins

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0130] The nucleotide sequence of the prolipase-bovine trypsinogen fusion protein is shown in SEQ ID 1 and the corresponding amino acid sequence is shown in SEQ ID 2.

[0131] Amplification of the prolipase gene fragment from the Rhizopus oryzae lipase / pPIC9K vector using high-fidelity PWO polymerase and the following primers:

[0132] PRORHILIPFP2=5'CTC GAG AAA AGA GAG GCT GAA GCT GTTCCT GTT TCT GGT AAA TC3'

[0133] PLBTRRP=5'TTG TCA TCG TCA TCG GCG CTG TTG GTA GATCCA GA 3'

[0134] The bovine trypsinogen gene was amplified from the bovine trypsinogen / TA vector using high-fidelity PWO polymerase and the following primers: The bovine trypsinogen gene was codon-optimized using the Software-based Entechelon Net.

[0135] PLBTRFP=5'CTA CCA ACA GCG CCG ATG ACG ATG ACA AGATTG TCG GA 3'

[0136] BTRPRP1=5'GCG GCC GCT TAG TTA GAC GCA ATT GTT TGCTTG 3'

[0137] These products were purified using Qiagen's Gel Extraction Kit. 2 µl each of these purified products were used as templ...

example 2

[0143] This product was subcloned into pPIC9K:

[0144] The PLBTR fragment was excised using Xhol and EcoRI sites and ligated into pPIC9K at the same sites. The ligation mixture was transformed into competent E. coli DH5α cells, and clones were selected on LB agar plates containing 100 μ / ml ampicillin. The clones obtained were screened using the boiling miniprep method. The presence of the insert was confirmed by releasing the insert digested with the restriction enzymes Xhol and EcoRI. The correct clone named PLBTR / pPIC9k was verified by restriction digest.

[0145] Transformation of Pichia pastoris GS115 strain with PLBTR / pPIC9K plasmid:

[0146] The PLBTR / pPIC9K vector was linearized using Sad and transformed into Pichia pastoris GS115 by electroporation following the method described in the Invitrogen manual. About 1200 clones were screened on G418 at 0.5 mg / ml. Forty-one clones were found to be resistant to 0.5 mg / ml of G418. These clones were placed on G418 at 2 mg...

example 3

[0155] Expression of Pichia codon-optimized prolipase-bovine trypsinogen (CPLBTR) in a homemade Pichia pastoris strain:

[0156] The synthetic gene of Pichia pastoris codon-optimized prolipase-bovine trypsinogen (CPLBTR) is represented in SEQ ID:3.

[0157] Cloning of trypsinogen in pMBL210.

[0158] 1. PCR amplification:

[0159] PCR amplification of lipase-bovine trypsinogen was performed using plasmid 0900098 Seq 3 pMA obtained from Geneart using primers CPLBTRFP and CPLBTRRP.

[0160] CPLBTRFP: 5’ACC TCG AGAAGA GAG TTC CAG T 3’

[0161] ·CPLBTRRP: 5’GGG AAT TCT TAG TTA GAA GCG ATA GTT TGC 3’

[0162] PCR reaction mix:

[0163] water

37μl

0900098 Seq 3 pMA,

1.5μl (50ng)

dNTP mix

5μl

CPLB TRFP

1μl (0.01μmol)

CPLB TRRP

1μl (0.01μmol)

10X Amplification High Fidelity Assay Buffer

5μl

amplified high-fidelity polymerase

0.5μl

total capacity

50μl

[0164] PCR conditions:

[016...

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Abstract

The present invention relates to novel Prolipase-Bovine trypsinogen (PLBTR) fusion proteins, the genes encoding them, and the production and uses thereof. More specifically, the present invention relates to methods of producing in optimal quantities PLBTR fusion proteins which comprise a heterologous polypeptide which is normally susceptible to autocatalytic activity. More particularly, the present invention relates to fusion proteins which comprise an heterologous polypeptide, such as a serine protease, fused to a lipase signal sequence, which can be expressed by recombinant host cells in desired amounts. The present invention further relates to polynucleotides encoding such fusion proteins, to expression vectors for expression of such fusion proteins, to host cells transformed with such polynucleotides / vectors, and to methods of generating such fusion proteins.

Description

technical field [0001] The present invention relates to novel prolipase-bovine trypsinogen (PLBTR) fusion proteins, genes encoding them, and products and applications thereof. More specifically, the present invention relates to methods for optimal production of PLBTR fusion proteins comprising heterologous polypeptides that are normally susceptible to autocatalytic activity. More specifically, the present invention relates to fusion proteins comprising a heterologous polypeptide, such as a serine protease fused to a lipase signal sequence, which can be expressed in desired amounts by recombinant host cells. The invention also relates to polynucleotides encoding such fusion proteins, to expression vectors for expressing such fusion proteins, to host cells transformed with such polynucleotides / vectors, and to methods of producing such fusion proteins. Background technique [0002] Trypsin is a high-value protease with many industrial and biomedical applications. The continuo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/81C12N15/57C12P21/06
CPCC12N9/20C12N9/6427C07K2319/00C12P21/06C12N9/6424C07K19/00C12N15/62C12N15/52C12N15/81
Inventor 纳加拉杰·戈文达帕南迪尼·纳塔拉杰桑贾伊·蒂瓦里帕萨·哈兹拉穆克什·巴布阿帕·帕塔勒戈库尔·约蒂拉曼克达纳斯·萨斯特里
Owner BIOCON LTD
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