A method for regulating protein expression based on N-terminal coding sequence modification

Abstract

The invention discloses a method for modifying and regulating protein expression based on an N-terminal coding sequence, and belongs to the technical fields of genetic engineering and enzyme engineering. The invention uses Bacillus subtilis as an expression host, and evaluates the most favorable nucleotide sequence for promoting gene expression in the synonymous mutation of the N-terminal coding region through a prediction model. By combining the first ten amino acid synonymous mutation libraries of NCS with superfolded green fluorescent protein (sfGFP), the fluorescence intensity of the protein in the library was measured, 172 representative samples were selected and identified by sequencing, and a prediction model was established using statistical methods. Using this model to optimize the pullulanase fused with the BlgS signal peptide, the extracellular enzyme activity of pullulanase can be increased to 2.67 times and reduced by 48% before the transformation, thus providing a rational transformation direction for the de novo design of the N-terminal gene , which is conducive to the simple regulation of gene expression.

Description

technical field [0001] The invention relates to a method for modifying and regulating protein expression based on an N-terminal coding sequence, and belongs to the technical fields of genetic engineering and enzyme engineering. Background technique [0002] Gene mutation is of great significance for changing the properties of proteins. Usually through mutations, mutation sequences with better properties can be found, thereby improving the application value of proteins. The synonymous mutation of the gene is a common mutation method, and the synonymous mutation of the gene can achieve a huge difference in expression. [0003] The currently commonly used method is to construct a synonymous mutation library, combined with high-throughput screening strategies, in order to find the best mutants. However, this method is time-consuming, labor-intensive, and highly specific, and cannot be used to guide the design of other genes. Although some studies have found that synthesizing a...

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Problems solved by technology

However, this method is time-consuming, labor-intensive, and highly specific, and cannot be used to guide the design of other genes

Although some studies have found that synthesizing a series of short peptides is beneficial to widely improve gene expression, this method will affect the enzyme activity, because these expression-promoting short peptides occupy the position of the signal peptide, thus not Suitable for extracellular proteins that require the addition of signal peptides

[0004] The existing methods for improving gene expression are often through the optimization of the untranslated region (5'UTR). However, when the 5'UTR module is already strong enough, it is difficult to continue to optimize and significantly increase the expression level

However, there are few studies on the N-terminal coding region (NCS)

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