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Method for producing recombinant human interleukin-21 by using Pichia pastoris

A technology for human interleukin and Pichia pastoris, which is applied in the field of bioengineering and can solve the problems of increased molecular weight and low expression level.

Inactive Publication Date: 2011-04-20
SHANGHAI GENON BIOENG +1
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0005] At present, hIL-21 is mainly produced through the prokaryotic expression system of Escherichia coli, but this system has the following disadvantages: (1) the expression level is not high, (2) it is expressed in cells, (3) it exists in the form of inclusion bodies, which needs to be denatured, Refolding operation
Japan's Asano and others also produced hIL-21 through the E. coli expression system, and the expression level can reach up to 200mg / L (Asano R, Kudo T, Makabe K, et al.Antitumor activity of interleukin-21 prepared by novel refolding procedure from inclusion bodies expressed in Escherichia coli. FEBS Lett, 2002, 528(1-3): 70-76.), but because the obtained hIL-21 has a His tag, the relative molecular weight increases, and it also exists as an inclusion body In Escherichia coli cells, tedious operations such as crushing the bacteria, denaturation and gradual renaturation are required

Method used

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  • Method for producing recombinant human interleukin-21 by using Pichia pastoris
  • Method for producing recombinant human interleukin-21 by using Pichia pastoris
  • Method for producing recombinant human interleukin-21 by using Pichia pastoris

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Construction of rhIL-21 engineering bacteria

[0063] 1. Acquisition and analysis of hIL-21 gene fragment sequence

[0064] First, the gene encoding hIL-21 was analyzed, and the results showed that the gene does not contain the sequence encoding the substrate of Pro-Glu-Ser-Thr, which activates proteolytic enzymes, nor does it contain the sequence encoding the gene that is susceptible to lysosomal cleavage. The sequence of X-Phe-X-Arg-Gln, Gln-Arg-X-Phe-X (X = any amino acid) of At the same time, considering that the codon preference of yeast is different from that of humans, the CAI value of hIL-21 coding gene expressed in Pichia pastoris is 0.74, which has the potential of encoding high-level expression protein (>0.6), and the CAI value is synonymous. A statistical measure of codon bias that can be used to predict protein expression levels. Therefore, the lymphocytes were directly isolated from the peripheral venous blood of healthy people, the total RNA of the cell...

Embodiment 2

[0081] Induced expression of hIL-21 in shake flasks

[0082] 1. Screen the engineering bacteria with the highest expression

[0083] A two-step culture method was adopted, and the expression level of each transformant was expressed in shake flasks. The example was to pick multi-copy integrated recombinant transformants and inoculate them in 10mL BMGY medium, and culture them with shaking at 28-30°C and 250r / min until the bacteria When the OD600 of the solution is 2-6, centrifuge to remove the supernatant, replace it with BMMY medium to continue culturing, add methanol every 24 hours to a final concentration of 0.5%, and collect the bacterial solution after continuous induction for 72 hours. The supernatant was collected after the bacterial solution was centrifuged, part of the supernatant was concentrated with trichloroacetic acid, and the relative molecular weight of rhIL-21 was verified to be 16kD by 15% SDS-PAGE electrophoresis. Using IL-21 polyclonal antibody to do Wester...

Embodiment 3

[0091] Detection of hIL-21

[0092] Using a fast protein liquid chromatography system ( ), the protein in the expression supernatant was purified using the cation exchange medium SPSepharose Fast Flow. Use ABIPROCISE TM 492cLC (GC320078) instrument detects the first 15 amino acids of the N-terminal of purified rhIL-21. The detection method is based on the protein N-terminal sequencing standard method (SCI-S-006). The first 15 amino acids of the rhIL-21 N-terminal sequence are: Acid-Valine-Glutamic Acid-Leucine-Glutamine-Aspartic Acid-Arginine-Histidine-Methionine-Isoleucine-Arginine-Methionine- Arginine-glutamine-leucine (YVELQ DRHMI RMRQL) further confirmed that the expression product was rhIL-21.

[0093] In addition, the results of biological activity experiments show that the expression product can stimulate the proliferation of human lymphocytes, and the stimulation degree is the same as that of the commercially available IL-21 standard product.

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Abstract

The invention provides a method for producing recombinant human interleukin-21 by using Pichia pastoris, in particular to a production method of the recombinant human interleukin-21 expressed by the Pichia pastoris, which comprises the following steps: firstly, the reverse transcription PCR (polymerase chain reaction) is carried out in lymphocytes of healthy people to obtain encoding genes of rhIL-21, and the encoding genes are fused in expressional regulatory elements of the Pichia pastoris to construct Pichia pastoris high-level-expression engineering bacteria; and secondly, the Pichia pastoris high-level-expression engineering bacteria are induced to produce a large number of recombinant human interleukins-21 by adding methanol. The Pichia pastoris is very easy to achieve high-density fermentation and has the characteristics of hypersecretion, so that a large number of recombinant human interleukins-21 can be industrially produced easily at low cost.

Description

technical field [0001] The invention relates to the field of bioengineering, and more specifically relates to a method for producing recombinant human interleukin-21 by Pichia pastoris. Background technique [0002] With the rise of genetic engineering pharmaceuticals, many natural human protein factors with medicinal value are produced on a large scale through recombinant technology. At present, about 50 kinds of genetically engineered protein drugs have been approved for marketing by various countries, which have benefited patients and also achieved huge economic benefits. For example: human growth hormone, human insulin, Epo, G-CSF, etc. [0003] Human interleukin 21 (Huamn Interleukin 21, hIL-21) is a type I cytokine discovered by Parrish Novak et al. in 2000, which is activated by CD4 + Produced by T cells, NKT cells, Tfh cells and Th17 cells, it has high homology with IL-2, IL-4 and IL-15, and belongs to the γc family members. IL-21 can specifically bind to interleu...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N1/19C12P21/02C12R1/84
Inventor 成国祥俞慧清李栋陈建泉刘思国
Owner SHANGHAI GENON BIOENG
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