Polypeptide gapM1 and its prepn process

Abstract

The present invention belongs to the field of biotechnology, and is especially one kind of polypeptide gapM1 and its preparation process. The polypeptide has the amino acid sequence of sequence 2. After the gapM1 is codon preference designed, optimized gapM1 gene segment is synthesized in PCR process, recombined with expression vector and transformed to corresponding expression cell to screen high expression engineering bacterium or engineering cell. After proliferation, collecting culture medium supernatant and separating purification, high purity target protein is obtained. The prepared polypeptide may be expressed in soluble activity form, and has high expression level, high yield, and the effects of lowering blood sugar, lowering blood fat, reducing weight, etc. The preparation process is superior to available technology, and has the advantages of simple production process and low production cost.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a novel polypeptide called gapM1 (globular domain of adipose abundant manuscript 1, gapM1) and a preparation method thereof. Specifically, the present invention relates to the amino acid sequence and gene sequence of gapM1, gene codon optimization (nonsense mutation), protein expression, protein purification and functional determination. Background technique [0002] apM1 (adipose abundant manuscript 1, apM1) is a plasma protein secreted by body cells such as adipocytes, which has important physiological functions such as lowering blood sugar, blood lipids and body weight. gapM1 is the globular domain of full-length apM1 and is the active domain of full-length apM1. Full-length apM1 and gapM1 exist simultaneously in vivo, and gapM1 is a product digested by protease in vivo from full-length apM1. The globular domain of full-length apM1 (gapM1) has 137 amino acid residues. It ...

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Problems solved by technology

Although the gapM1 expressed by this method exists in an active form, the expression level of the CHO eukaryotic expression system is very low; the production cycle is very long, dozens of times that of Escherichia coli, yeast and other expression systems; at the same time, the production cost of this system is extremely high, The production process is difficult to control; the addition of protease in the same enzymatic digestion process is not conducive to downstream purification

3), the patent CN1380304 expresses gapM1 directly using the E. coli system. Although this method omits the process of protease digestion, gapM1 exists as an inclusion body in an inactive form, and there is no globular domain after the amino terminal due to two The characteristics of the secondary structure (full β-sheet structure), it is more difficult to renature in vitro than the full-length apM1, and the yield is the lowest among the above methods; at the same time, gapM1, which is difficult to renature, is very soluble in most steps of the production process. Poor, which brings great inconvenience to the production process

Due to various defects in the above-mentioned known technologies, the phase III clinical trial of gapM1 in foreign countries was finally terminated due to the difficulty in preparing gapM1 and failing to meet the needs of the experiment.

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