Preparation of recombinant human hemoglobin with pichia stipitis

Abstract

The invention provides a method for preparing functional recombinant human hemoglobin. The method comprises the following steps of culturing yeast Pichia in a culture medium and recovering human hemoglobin from an obtained culture, wherein the yeast Pichia is transformed from an expression vector of encoded human hemoglobin alpha chain DNA which is contained in the yeast Pichia under the regulation of a promoter capable of playing a role, as well as an expression vector of encoded human hemoglobin beta chain DNA which is contained in the yeast Pichia under the regulation of another promoter capable of playing a role. The invention also provides an expression vector. From the upstream side in a transcription direction, the expression vector successively comprises one ethanol oxidase 1 promoter, the encoded human hemoglobin beta chain DNA, one terminator capable of playing a role in the yeast Pichia, one ethanol oxidase 1 promoter, the encoded human hemoglobin alpha chain DNA and one terminator capable of playing a role in the yeast Pichia.

Description

technical field [0001] The invention relates to the preparation of recombinant human hemoglobin and the expression carrier used therein. In more detail, the present invention relates to a method for preparing human hemoglobin using yeast of the genus Pichia, and an expression cassette comprising a DNA encoding a human hemoglobin β chain and a DNA encoding a human hemoglobin α chain connected in tandem The expression vector of the expression cassette. Background technique [0002] Hemoglobin is a protein that exists in high concentrations in red blood cells and has the function of carrying various gas molecules represented by oxygen. Its structure is a heterotetramer composed of two α-chains and β-chains respectively. [0003] As preparations utilizing hemoglobin, erythrocyte substitutes have been developed. So far, people have designed the modified body of hemoglobin—intramolecular bridging type, overlapping type, polymer binding type and hemoglobin small cell body that e...

AI Technical Summary

Problems solved by technology

However, there are such problems in this method: (1) Enzymes such as methionine aminopeptidase are expressed together with hemoglobin, and an extra first methionine is added to the N-terminus, which is different from the sequence of the natural primary structure (2) In order to synthesize heme, aminolevulinic acid, etc. must be added to the culture medium

[0008] However, when the heterologous protein of interest is combined with the host cell (phase compatibility), the expression efficiency of the heterologous protein is often low

In particular, hetero-oligomeric proteins such as hemoglobin must be aware of various conditions such as the balance of expression between the subunits and the formation of oligomers, and it is not easy to obtain the expected high expression

In fact, there are extremely limited examples of Pichia yeasts being used for the industrial production of human proteins

Images