Method for preparing recombinant serratia marcescens nuclease

Abstract

The invention provides a method for preparing recombinant serratia marcescens nuclease. The method comprises the following steps: carrying out fusion expression on coding nucleic acid of the serratia marcescens nuclease and coding nucleic acid of a mutant SUMO3 tag, and separating to obtain the serratia marcescens nuclease. The yield of the protein is improved by optimizing prokaryotic expression; through mutation modification of SUMO3, the renaturation rate and uniformity of the recombinant serratia marcescens nuclease are improved. Meanwhile, the purification steps are simplified, the production cost is reduced, and the method is suitable for large-scale industrial production of the recombinant serratia marcescens nuclease.

Description

technical field [0001] The invention belongs to the field of biomedicine; more specifically, it relates to a method for preparing recombinant Serratia marcescens nuclease. Background technique [0002] Nucleases are enzymes that cleave phosphodiester bonds between nucleotide subunits of nucleic acids. Plays an important role in many aspects of genetic machinery, including participation in avoiding mutations (DNA repair, replication and recombination), removal of excess nucleotides and phosphates produced during growth and metabolism, defense against foreign nucleic acid molecules, programmed cell death and infection; in the food industry, nucleases can be used to produce nucleic acid flavor enhancers, such as guanosine monophosphate, inosine monophosphate, etc.; in the pharmaceutical industry, nucleases are used to produce other nucleotides, vaccines and gene therapy products. [0003] Nucleases can be divided into two categories according to their substrate specificity: su...

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Problems solved by technology

[0005] In the existing method, although the recombinant Serratia marcescens nuclease is secreted and expressed in Escherichia coli, still about 50% of the recombinant Serratia marcescens nuclease cannot be secreted into the culture medium (see the invention for details Patent US5173418A), so that its yield is severely limited, and the recombinant Serratia marcescens nuclease remaining in the cell has the ability to hydrolyze the E. coli genome, damage the E. coli genome, and further limit the recombinant Serratia marcescens nucleic acid Enzyme production, so the yield of recombinant Serratia marcescens nuclease is low, even if the method of high-density bacterial fermentation is adopted, it is still necessary to process tens to hundreds of liters of medium supernatant, increasing the recombinant Serratia marcescens Production time and cost of Bacillus nuclease

In addition, for example, in the scheme of US5173418A, the constructed plasmid does not have a purification tag, so fast and efficient affinity chromatography purification methods cannot be used, and only multi-step purification methods such as ammonium sulfate precipitation, ion exchange chromatography, and size exclusion chromatography can be used. , resulting in cumbersome purification steps and long production time

In some other existing expression methods, although the purification efficiency is improved by introducing tags at the N-terminal and C-terminal of the recombinant protein, the tags cannot be completely excised. The Serratia marcescens nuclease obtained by recombinant production has the same There are differences in the amino acid sequences of the nucleases

[0006] In summary, the existing invention patents for the preparation of recombinant Serratia marcescens nuclease have problems such as low protein yield, protein inhomogeneity, cumbersome purification steps, long production time and high production cost.

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