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Method for preparing human serum albumin-human parathyroid hormone

A technology of parathyroid hormone and human serum albumin, applied in peptide/protein components, chemical instruments and methods, pharmaceutical formulations, etc.

Active Publication Date: 2013-09-18
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But more and more evidence shows that yapsins deficiency can significantly improve the degradation of some specific proteins in Saccharomyces cerevisiae or Kluyveromyces lactis

Method used

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  • Method for preparing human serum albumin-human parathyroid hormone
  • Method for preparing human serum albumin-human parathyroid hormone
  • Method for preparing human serum albumin-human parathyroid hormone

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Amplification of the homology arm at the 5' end of YPS1 and the homology arm at the 3' end

[0022] Obtain the 5' end homology arm fragment and the 3' end homology arm fragment of YPS1 sequence from yeast genome by PCR method, used primer YPS1_N up (SEQ ID NO.1), YPS1_N dn (SEQ ID NO.2) and YPS1_C up (SEQ ID NO.3) and YPS1_C dn (SEQ ID NO.4) were synthesized with an oligonucleotide synthesizer. The upstream and downstream primers for the amplification of the homology arm at the 5' end are respectively introduced into the PstI and SalI sites and the protective bases, and the upstream and downstream primers for the amplification of the 3' homology arms are respectively introduced into the BglII and PstI sites and the protective bases base, the endonuclease recognition sequence is underlined.

[0023] YPS1_N up: 5'-aagc ctgcag agctccattg cgccaacccc-3'

[0024] YPS1_N dn: 5'-cggc gtcgac aatctggctg agcggaaagt ttga-3'

[0025] YPS1_C up: 5'-ccc agatct c a...

Embodiment 2

[0028] Example 2: Construction of recombinant vector pPICZαB-YPS1Δ

[0029] After electrophoresis purification of the 3' homology arm PCR product obtained in Example 1, the target band was recovered and purified with a DNA fragment recovery kit. The purified 3' homology arm PCR product was digested with BglII / PstI and recovered by electrophoresis; the plasmid pPICZαB was also digested with BglII / PstI, and the linearized fragment was recovered by electrophoresis. The digested 3' homology arm and the vector pPICZαB were mixed in an appropriate ratio, and ligated with T4 ligase overnight in a water bath at 16°C to form pPICZαB-C. After transformation into DH5α, spread on LB agar plates containing 25 μg / mL Zeocin, and culture overnight at 37°C. Select several clones on the plate and inoculate them into 5 mL LB liquid medium containing 25 μg / mL Zeocin, and culture overnight at 37°C. The positive clone pPICZαB-C / DH5α was obtained by PCR verification of YPS1_C up / YPS1_C dn with pri...

Embodiment 3

[0031] Example 3: Homologous recombination method to knock out the YPS1 gene in the host SMD1168

[0032] The Pichia host SMD1168 is a protease A-deficient host. Therefore, on the basis of SMD1168, the aspartate hydrolase 1 gene can be further knocked out to obtain a Pichia host with a double knockout of protease A and yapsin 1.

[0033] After linearizing pPICZαB-YPS1Δ with PstI, it was transformed into Pichia pastoris SMD1168 by electroporation (the yeast expression kit was provided by Invitrogen Corp. / mL Zeocin and 1M sorbitol on YPD agar plate (1% yeast extract, 2% peptone, 2% glucose, 1M sorbitol, 2% agarose), culture at 30°C for 3-5 days. Pick several clones on the plate and inoculate them in 5 mL of YPD liquid medium containing 50 μg / mL Zeocin, and culture overnight at 30°C. PCR verification of positive clones, see image 3 , Figure 4 : Design a pair of negative primers (yps1 negative up (SEQ ID NO.5), yps1 negative dn (SEQ ID NO.6)), a pair of positive primers (yps...

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Abstract

The invention provides a method for preparing human serum albumin-human parathyroid hormone. By means of transforming a yeast host SMD1168, yapson1 coded sequences on the SMD 1168 can be knocked out. On the basis of the SMD 1168, coded gene YPS1 of yapson1 is further knocked out, namely double knockouts of a proteinase A and yapsin1 are achieved, and the transformed host is named as SMD1168yps1 delta. By the aid of the transformed host, a recombination expression of fusion proteins of human serum albumin-human parathyroid hormone (1-34) is optimized, so that the degradation of interest proteins in the expressing process can be reduced, and downstream mass production costs and difficulties of separation and purification are lowered. According to the method, the optimized the human serum albumin-human parathyroid hormone (1-34) fusion proteins can be applied to prepare parathyroid hormone (PTH) (1-34) medicines which are used for treating osteoporosis.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, it relates to the construction of genetically engineered bacteria with double knockout of Pichia protease A and yapsin 1, and using the host for the corresponding proteolytic enzyme-sensitive heterologous protein—human serum Recombinant expression of albumin-human parathyroid hormone (1-34) fusion protein. technical background [0002] The Pichia pastors expression system is one of the best and most widely used exogenous protein expression systems. It not only overcomes the shortcomings of the E. coli expression system that cannot express proteins with complex structures, the expressed proteins often form insoluble inclusion bodies, many background proteins, and low expression yield, but also makes up for the complex operation and low expression level of mammalian cells and insect cell expression systems. 1. The industrial production cost is expensive, but it also has advantages that ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12N15/81C07K19/00A61K38/29A61K47/48A61P19/10
Inventor 吴敏沈其王芙蓉徐迎春陈枢青
Owner ZHEJIANG UNIV
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