Protein engineering method based on isopeptide bond scanning and application of method to protein engineering

Abstract

The invention relates to an isopeptide bond scanning method and an application thereof to protein engineering. According to the method, a microorganism is used as a host, a SpyTag and SpyCatcher system can be self-catalyzed to form isopeptide bonds, a SpyTag and SpyCatcher randomly replaces an optional amino acid of a certain target protein peptide chain or is inserted into a coded sequence of a peptide chain obtained at an optional position of the target protein peptide chain and placed at a proper promoter or terminator, an expression cassette is built, and transcription and translation arerealized under control of the host to obtain a target protein product set with the isopeptide bonds spontaneously formed among different amino acids of a polypeptide chain. Different modified proteinscan be acquired by the aid of a proper screening method. The isopeptide bond scanning method is developed for recombinant protein modification and used for rapid artificial evolution of the proteins.

Description

technical field [0001] The invention belongs to the field of protein engineering, and relates to the formation of isopeptide bonds, in particular to the formation of isopeptide bonds by molecular self-catalysis mediated by the SpyTag-SpyCatcher system, in particular to a protein engineering method based on isopeptide bond scanning and its application in applications in protein engineering. Background technique [0002] Generally, protein engineering focuses on solving the following two problems: [0003] 1. The problem of protein thermodynamic stability. [0004] 2. High efficiency of protein function dynamics. [0005] At present, there is room for improvement in the following two aspects of protein engineering technology: [0006] 1. The limitation of structure-activity relationship research, the efficiency of protein modification by site-directed mutagenesis is not high [0007] Nucleotide-based directed evolution causes meaningless molecular diversity due to codon de...

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Problems solved by technology

[0003] 1. The problem of protein thermodynamic stability

[0004] 2. High efficiency of protein function dynamics

[0008] 2. Fixed-point chemical modification of peptides or proteins has problems such as high production costs and poor control of product quality uniformity

But this process also has obvious limitations: First, it is necessary to ensure that sufficient biodiversity is generated, which requires protein (nucleic acid) molecules to have sufficient length; second, because most of them are negative mutations, the screening work relies on Qualcomm Finally, due to the degeneracy of codons and the generally small influence of single amino acid mutations in enzyme proteins, the probability of obtaining good enzyme proteins by random nucleotide mutations is further reduced

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