Unlock instant, AI-driven research and patent intelligence for your innovation.

Method for large-scale purification of recombinant proteins in laboratory

A recombinant protein and large-scale technology, applied in the biological field, can solve the problems of long purification process, time-consuming, laborious, low efficiency, etc., and achieve the effect of shortening the purification process time, simplifying the process, and reducing the workload

Active Publication Date: 2015-06-03
VIVA BIOTECH
View PDF3 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Such a fixed large-scale purification mode has existed in many laboratories for a long time, and such a method is extremely inefficient for companies pursuing profit maximization
According to the analysis, this traditional method has great defects. There are two main points: 1. The purification process takes a long time
2. It requires a lot of manpower, and there is no way to complete it independently in limited working hours
Such a time-consuming and labor-intensive traditional large-scale purification mode is fatal for purifying proteins that are easily degraded and require high enzyme activity
[0004] In recent years, the research on many target proteins has become more and more popular, and there are more and more interdisciplinary researches. The demand for research-type proteins continues to expand, but the quality requirements are more stringent. It is obvious that the use of traditional large-scale purification methods to obtain high-quality proteins cannot meet this need

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Take 700g of protein A bacteria, put 5L of buffer in a beaker, and use an electric mixer to completely dissolve it (stirring with a traditional rotor and a glass rod takes 3 hours for one person to operate, and only 30 minutes for an electric mixer), and the dissolved sample is processed by a high-pressure homogenizer. Cell crushing (traditional small-volume ultrasound requires one machine and one person to operate for 17 hours, while high-pressure homogenization requires one machine and one person to operate for only 1 hour), the crushed samples are placed in a 500ml centrifuge cup, and a high-speed centrifuge is used to Centrifuge at 9000rpm for 30 minutes, replace the centrifuged supernatant with a centrifuge cup and centrifuge again (this centrifugation process takes about 2.5 hours for a centrifuge with a 6-hole rotor, while the traditional method is to use a 50ml centrifuge tube to centrifuge, the same centrifuge with 8 holes The rotor takes about 15 hours). Pour ...

Embodiment 2

[0032] Take 1300g of protein B bacteria, put 10L of buffer solution in a beaker, and dissolve it thoroughly with an electric mixer (traditional rotor and glass rod stirring, two people need 3 hours; electric mixer, one person only needs 50 minutes), and the dissolved sample is passed through high pressure The homogenizer is used for cell crushing (traditional small-volume ultrasound requires 4 machines and two people to operate for 8 hours, while the high-pressure homogenizer only needs 1 hour for one machine and one person to operate), and the crushed samples are placed in a 500ml centrifuge cup. Use a high-speed centrifuge to centrifuge at 8000rpm for 30min, replace the centrifuged supernatant with a centrifuge cup and centrifuge again (this centrifugation process requires two centrifuges with 6-hole rotors, and one person needs about 2 hours to operate; while the traditional method is to centrifuge with a 50ml centrifuge tube, Similarly, the 8-hole rotors of two centrifuges ...

Embodiment 3

[0034] A method for large-scale purification of recombinant protein in the laboratory, the method comprises the following steps:

[0035] (1) Bacteria melting

[0036] Put 100g of frozen bacteria in a beaker, add 700ml tris buffer solution, put the beaker with bacteria in the stirring paddle of an electric mixer and mix well, the stirring speed is 300 rpm , the stirring time is 2 hours;

[0037] (2) High-pressure homogenate lysed bacteria

[0038] The dissolved large-volume bacteria are directly fully lysed with a high-pressure homogenizer; the pressure of the high-pressure homogenizer is 500 bar, and the lysis is performed for 2 cycles;

[0039] (3) centrifugal

[0040] Pack the lysed bacteria into 500ml centrifuge cups, balance them in pairs and place them in a pre-cooled centrifuge for centrifugation. Replace the centrifuged supernatant with a new centrifuge cup and centrifuge again; the speed of the centrifuge is 7000rpm. The centrifugation time is 30min;

[0041] (4)...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to a method for large-scale purification and recombinant proteins in a laboratory. The method comprises the following steps: (1) placing frozen thalli into a beaker, adding a denaturing lysis buffer according to a certain proportion and sufficiently and uniformly mixing; (2) directly and sufficiently performing pyrolysis on the well-dissolved large-volume thalli by a high-pressure homogenizing instrument; (3) placing the well-pyrolyzed thalli into a precooled centrifugal machine to carry out centrifugation, and placing a well-centrifuged supernatant into a new centrifuge cup to carry out centrifugation again; (4) pouring clear supernatant into the beaker, then adding a filler which is balanced in advance into the beaker, and placing the beaker on a constant-temperature magnetic stirrer to stir for 2 to 3 hours in a refrigerator with a temperature of 4 DEG C; and (5) after stirring incubation, standing for 30 min, pouring out supernatant, performing low-speed centrifugation on a residual sample comprising the filler, removing the supernatant, suspending the centrifuged filler by the denaturing lysis buffer to directly place the filler into a chromatographic column and eluting the target proteins according to different purifying fillers by corresponding denaturing lysis buffers. The method has the advantages of labor saving, controllable quality, low cost and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a method for large-scale purification of recombinant protein in a laboratory. Background technique [0002] At present, the large-scale purification process of recombinant proteins in laboratories of most scientific research institutions, CRO companies or universities and other institutions is generally divided into the following points: 1. Thawing bacteria (put the frozen bacteria in a beaker, and add the cracking Buffer trimethylol base aminomethane buffer solution, place the beaker on a magnetic stirrer and stir with a magnetic rotor to fully mix). 2. Ultrasound (distribute the dissolved bacteria into small flasks, the bacteria liquid in each beaker is generally less than 150ml, place the beaker in a vessel containing an ice bath, and then place the ultrasonic probe in a suitable position of the bacteria, Finally, set the parameters of the ultrasonic instrument, and then start the u...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/22
Inventor 薛飞谭恩旺田为宇尚建强彭金河樊盼盼赵岩龙
Owner VIVA BIOTECH