Method for efficiently expressing DNA polymerase by using pichia pastoris

Abstract

The invention provides a method for expressing a DNA polymerase from Thermococcus kodakarensis strain by using pichia pastoris, which comprises the following steps of: 1) cloning the DNA polymerase gene to a pichia pastoris secretion type expression vector to obtain pichia pastoris recombinant expression plasmid; 2) converting the recombinant expression plasmid into pichia pastoris host cell GS115 by electric shock, and identifying and screening a positive clone through an MD panel and a PCR; and 3) performing induction expression on the selected positive clone by using 0.5 percent methanol. The method can be used for expressing the DNA polymerase gene from the Thermococcus kodakarensis strain. Through verification, compared with an Escherichia coli expression system, the method can improve the performance such as the heat stability and the like of the DNA polymerase obviously, reduce the production cost, shorten the process flow and simplify purifying steps.

Description

technical field [0001] The present invention relates to the induced expression technology of exogenous genes, especially a method for expressing DNA polymerase by using Pichia pastoris, which is a new idea, new approach and new method for the expression and production of DNA polymerase. Background technique [0002] Polymerase chain reaction (polymerase chain reaction, PCR) is one of the most important basic research methods in modern molecular biology. This technology is widely used in the fields of molecular biology, medicine and other related disciplines because of its advantages of simple operation, rapidity, high sensitivity and strong specificity. [0003] As one of the important tools in the polymerase chain reaction, DNA polymerase plays a vital role in the PCR process. Until 1988, Rand ll K Saiki et al. (Saiki R K, Gelfand D H, Stoffel S, ScharfS, et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 1988, 239: 487-491) ...

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Problems solved by technology

Therefore, many studies at home and abroad express the genes encoding extremases in heterologous common host bacteria (such as Escherichia coli). Most of the proteins expressed by this method can maintain the original thermal stability. Folding is not hydrolyzed by the host protease, so it can be purified by removing the host protein by heat denaturation method, and the enzyme yield is also improved. It is a relatively economical method, which promotes the wide application of PCR technology to a certain extent, and Saved the PCR cost, but still faced various problems such as difficult purification and higher cost in this process ( Baneyx Recombinant protein expression in Escherichia coli Current Opinion in Biotechnology Volume 10, Issue 5, 1 October 1999, Pages 411-421)

For example, if the culture temperature is too high, the growth rate is too fast or the growth is excessive, most of the target proteins expressed by foreign genes are likely to form inclusion bodies, which requires conversion into soluble proteins. This step is not only cumbersome but also reduces enzyme activity.

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