Protein synthesis screening method

Abstract

The invention discloses a protein synthesis screening method. The invention further discloses a protein screening chip which comprises a liquid flow layer, a pneumatic control layer and an elastic film, and the elastic film is arranged between the liquid flow layer and the pneumatic control layer. The screening method comprises the steps: carrying out a ligation reaction on a to-be-screened N-terminal sequence fragment, a target gene fragment and a ligation mixed solution in a circulating liquid flow pipeline; inputting an amplification mixed liquid into a circulating liquid flow pipeline, and carrying out an amplification reaction; and inputting a cell-free protein expression mixed liquid into the circulating liquid flow pipeline, carrying out cell-free protein expression reaction, and then carrying out enrichment and purification treatment to obtain the target protein. According to the rapid and convenient protein translation initiation region screening method, target gene template optimization, amplification, protein expression, purification and detection are integrated, operation is easy and convenient, and screening of an optimal carrier of the target protein can be completed within one day so as to improve the yield of the target protein.

Description

technical field [0001] The present invention relates to a protein translation initiation region screening method, in particular to a fast and convenient protein translation initiation region screening method to increase the yield of a target protein, and a corresponding high-throughput protein screening chip. Background technique [0002] Proteins are of great scientific and practical significance as potential therapeutic biologics, drug targets, and biocatalysts. At present, the production of recombinant proteins still mostly uses living cells as factories. The gene sequence of the target protein is inserted into a suitable expression vector and transferred into living cells to express and produce the protein. The existing technology has achieved the successful expression and production of many proteins. However, many important proteins are still difficult to express effectively due to various reasons, such as low-expression proteins, insoluble proteins, and cytotoxic prote...

AI Technical Summary

Problems solved by technology

Since the selection of the most suitable expression vector requires complex biological experiments such as transfer into competent cells, sequencing, plasmid extraction, transfer into new competent cells for expression, protein purification and identification, etc., it requires a lot of manpower, material resources and time

Among them, it takes 1 day to construct the expression vector; transfer to competent cells, culture on the plate overnight and pick the clones and send them to the sequencing company for sequencing verification on the next day. It takes at least 2 days to get the sequencing results and the results are correct. However, in actual operation, due to different vector constructions The difficulty is different, and it will probably take longer; after the sequencing is correct, it will take 1 day to transfer the plasmid into a new expression host and culture it overnight, 1 day to induce expression, and 1 day to purify and identify the protein. In the most ideal state Under this condition, it takes at least 6 days to identify the expression of a new protein

Aiming at the complexity of constructing vectors, the Gateway system developed by Invitrogen can enable efficient cloning of genes into multiple expression vectors, without the need for more complicated subcloning techniques after the construction of entry vectors; however, the vectors provided by Gateway technology are relatively fixed , autonomous transformation still needs to repeat the above complex steps of vector construction, and the enzymes and vectors required by Gateway are expensive and difficult to be widely used

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