Small peptides refer to those with less than 100 amino acids in length. In recent years, small peptides were widely used in the fields of medicine, disease treatment, molecule vaccine, etc., which include surface antigens, disease diagnosis, and treatment of AIDS and cancers. With the fast development of biotechnology, more and more small peptides were discovered. Accordingly, a large amount of peptides are needed to meet the requirement in a variety of industries such as functional research, clinical experiment, and disease treatment. Generally, chemical synthesis is the main route to produce peptides with less than 40 amino acids in length. During the process of chemical synthesis, due to the existence of certain incomplete chemical reactions and chemical modifications, even peptides with less than 40 amino acids are difficult to be synthesized (Dobeli, et al., 1998 Protein Expression & Purification, Vol. 12:404-414). Therefore, it became advantageous and necessary to utilize bio-system to produce peptides.
In 1950s, bacteria were used as bioreactors to produce pharmaceutical products. However, because bacteria are prokaryotes, which do not possess the processing system of eukaryote, its application are seriously limited for some proteins whose bioactivities rely on protein modification. As the second-generation bioreactor, yeast has come into use in the production of medicine product since 1970s. However, the problems of low yield and incomplete modification/processing seriously limited the extensive use of yeast. The third-generation bioreactor utilized higher plant and animal cells. Presently, eukaryote bioreactors are categorized into animal bioreactors and plant bioreactors, and animal bioreactor further includes cell culture and transgenic animal. Currently, major antibodies for pharmaceutical use are produced by CHO (or mice) cell line culture. The studies of transgenic animals mainly focus on the expression of recombinant protein in breast cells of transgenic cow or egg albumin. However, the problems of animal pathogens contamination, high cost, and high investment requirement severely limit their use. It is estimated that the maximum productive capacity of monoclonal antibody is about 1,000 kg per year worldwide. To obtain another 1,000 kg, 40 billion US dollars of investment and more that ten years of time are further needed. All these data indicate the current productive system and productive capacity of the recombinant protein is far from enough to meet market requirement. Accordingly, a highly efficient and safe expression system is needed to satisfy the huge market demand of small peptide production.
In most cases, peptides with at least 80 amino acids are needed for recombinant protein expression. Even in such a case, the expression level is rather low. Therefore, a major way to improve the expression level of the peptide is to use fusion protein strategy. Up to now, the studies on fusion protein expression systems are mainly suitable for Escherichia coli (E. coli) and yeast systems. For instance, maltose binding protein (MBP), FLAG (Einhauer et al. 2001 J. Biochem. Biophys. Methods, Vol. 49:455-465) and glutathione (GST) (Papaioannou et al. 2002 Protein Expression & Purification, Vol. 13:462-466) were used as fusion partners in E. coli and yeast. Although some fusion protein expression systems have been commercialized, they were merely used in basic researches in, labs. Studies on the expression of peptides in plant expression system are relatively new and have not achieved much progress so far. Recently, it has been reported that the fusion carrier of disulfide bond dismutase (PDI) and green fluorescence protein (GFP) was used to express peptide. Unfortunately, the expression level was quite low. On the other hand, many peptides have been successfully expressed in Escherichia coli and yeast systems, there existed an obvious risk of being contaminated by pathogens from hosts. Moreover, the problems of low expression level and formation of insoluble inclusion bodies in E.coli cells, and higher molecular weight of the fusion partners used in prokaryotes cause troubles to downstream processing and therefore are not suitable to be used in eukaryotes, especially higher plant cells. Though plant cells have been used to express peptide, the low expression level has always been a bottleneck problem in the researches.
Due to the defects and limitations mentioned above, to develop fusion protein expression vectors in higher plants becomes increasingly important. Using higher plant as bioreactor has the advantages of low cost, high level expression, easy to scale up, free of pathogen contamination, etc, making it a promising candidate for future peptide production. So far, due to the problems of relatively high molecular weight, lack of cell organelle transportation signals, etc., prokaryotes fusion expression systems are not suitable for expressions in higher plant cells. Therefore, it is of significance to explore and develop small peptide expression system that is suitable for higher plants. Using rice storage protein as fusion carrier, Ventria Bioscience Inc. in USA has successfully expressed small peptides. Nevertheless, though higher level expression was achieved by the company with the use of globulin as fusion protein carrier, its application was limited in many aspects since the use of globulin to express peptide caused solubility problems. Other than storage proteins, another two proteins massively expressed in rice endosperm are endoplasmic reticulum biding protein (BiP) and protein disulfide bond dismutase (PDI), both of which are stored in protein body I. The C-terminus end of Bip protein has molecular chaperone activity, facilitating the correct folding of protein into functional protein conformation. Using C-terminus of Bip protein as fusion carrier can not only accumulate the fusion protein inside protein bodies (similar to the accumulation of the storage protein in protein bodies), but also increase the solubility of the fusion protein, thus overcoming the problem of insolubility resulting from using storage protein as fusion carrier in conventional methods. Another non-storage protein that highly expresses in rice or wheat endosperm is protein disulfide isomerase (PDI). PDI has two functions. One is disulfide dismutase activity at N terminus, and the other is C-terminal possesses molecular chaperone activity. Accordingly, using its C terminal as fusion protein carrier can also achieve the purposes of improving both the expression and the solubility of the protein. By using C-terminal of non-storage protein PDI and Bip as fusion carrier which are expressed specifically in endosperm, the present invention can not only improve protein expression level but also overcome the solubility problem existed in other international patents where storage proteins were used as fusion proteins, thus conferring innovativeness and patentability to the present invention.
Insulin-like growth factor (IGFs) is one of the most important growth factors involved in various types of proliferations and metabolisms. It not only takes an important role in the growth of human skeleton, but also facilitates the maturation of relevant cells and associates with wound healing. IGF-1 is a single strand peptide with 70 amino acids, with 3 disulfide bonds and no glycosylation site (De Bree, et al. 1998 Protein Expression & Purification, Vol. 13:319-325). Based on the analysis of the position of the recognizable disulfide bond, it is believed that the secondary structure of IGF-1 could be similar to that of insulin, both of which have same conservative glycines in same positions and have similar nonpolar amino acid residue core. IGF-1 is widely used in clinics. Recombinant human IGF-1 (rhIGF-1) and its complex have been effectively used in treating growth hormone insensitivity syndrome (GHIS), which includes GH receptor deficiency, IGF gene deficiency, and block of signal transduction path of growth hormone. Moreover, IGF-1 has been used to treat patients suffered from type I or type II diabetes or patients with severe insulin resistant symptoms. With the administration of rhIGF-1, the symptoms were greatly relieved. rhIGF-1 or its complex rhIGF-I/IGFBP-3 can be further used to treat chronic inflammation, nutrition disorder, and other conditions such as Crohn's disease (also called segmental enteritis), juvenile chronic arthritis, bladder/gall bladder fibrosis, etc. Relevant studies on the pharmacodynamics of IGFs are very limited, however, the shortage of IGFs supply is believed to be one of major problems (Savage, et al. 2005 Edocr. Development. Vol. 9:100-106).