Background

Diabetes is a chronic non-communicable lifelong disease that threatens the health of people around the world. Type Ⅱ diabetes (T2D) accounts for over 90% of the total diabetes patients. According to the report of experts from the World Health Organization, the 21st century will be a world of diabetes epidemics period. ^[1.] The main clinical manifestation of T2D characterizes by a dysfunctional fasting blood glucose that reaches 7.0 mmol/L or higher, and insulin resistance caused by hyperglycemia. The most typical symptoms of the disease is "three more and one less", that is, more drinking, more urinating, more eating, and weight loss in a short period. During the progression of T2D, there are also accompanying by some symptoms, such as fatigue, malaise, memory loss, blurred vision, numbness of limbs, etc. Although many drugs have been approved to control blood glucose level in patients to some extent, they are unable to cure the disease. Inversely, the associated side effects caused by longtime administration of drugs pose serious challenges for doctors and patients.

Problem

So, what is the real cause of T2D? In fact, there are two main reasons. For the first, the functional defect of pancreatic islet β cells resulting in an insufficient insulin secretion, which makes an increase in blood glucose. The second is that insulin secretion is normal but the body is insensitive to insulin, which is called insulin resistance. Therefore, it is necessary to develop new adaptable oral hypoglycemic drugs to control such diseases.

P37 (hereinafter referred to as vglycin), derived from soybean, is a novel orally administrated hypoglycemic peptide drug, that can improve blood glucose and blood lipid disorders in diabetic mice. Long-term administration of P37 can bring no any side effects in animal. Currently, P37 are being developed as a new drug for the prevention and treatment of metabolic syndrome such as diabetes and hyperlipidemia.^[2.] However, direct extraction from soybean has some problems such as low yield, long cycle and complicated process, which is not conducive to large-scale production.

Hubei University of Technology group proposed using synthetic biology to produce the hypoglycemic peptide P37 to overcome the low yield extracted from soybeans. Large amount of expression of hypoglycemic peptide P37 in Pichia pastoris is beneficial to reducing cost and easily expanding manufactory technique.

Pichia pastoris

In order to select proper expression vector, our team tried to collect information on various microorganisms. Ultimately, we chose Pichia pastoris as the expression host. Pichia pastoris is a kind of yeast which can use methanol as the only carbon source and energy source. Compared with other expression systems, Pichia pastoris has obvious advantages in the processing, exocrine, posttranslational modification and glycosylation modification of expression products. As one of the most commonly used protein expression system, Pichia pastoris is widely recognized as a prominent host in molecular biology for the purpose of generating recombinant proteins. The advantages of utilizing the pichia pastoris system for protein production encompass the proper folding process occurring within the endoplasmic reticulum.

His-tag

His-tag is a kind of tag often used in protein recombination technology. It contains six histidine, characterized by small molecular weight less than 0.84 KD, but does not change the biological structure and solubility of protein. More importantly, it makes the protein purification extremely convenient. According to the principle that the imidazole ring on histidine can bind to divalent Ni ions, which is convenient for further purification and detection. Based on this theory, we put His-tag at the carbon end of vglycin chain for the convenience of peptide purification.

Acid sensitive sites

In order to increase the expression yield of the objective peptide and facilitate the later purification, we concatenated three coding vglycin genes in the process of designing plasmid. An acid-sensitive site, Asp-Pro, was inserted between each vglycin peptide. When the product is enriched at a later stage, they will be placed in an acid solution at a pH~ 5, where the acid-sensitive site disconnects automatically and three separated vglycin molecules are formed.