Expression And Characterization Of Human Type Ⅲ Collagen In Pichia Pastoris

Collagen is an abundant structural protein in all animals. In humans, collagen comprises one third of the total protein, accounts for three quarters of the dry weight of skin. Collagen is also widespread in animal muscle sheath, tendon, cartilage, ligaments and skin, it is the main ingredient of ligaments and tendons, as it has a strong ability to assert. In the cytoplasmic matrix, collagen plays a supporting role to maintain the internal environment. It is reported that there are28different types of collagen, the type of collagen is different as they distribute in different tissue. Type Ⅲ collagen is the most prevalent component of blood vessels, skin and gut. Collagen application field is widely. Using the traditional collagen production method is far more to meet the demand. And extracting from animals, viruses or using chemical synthesis method to produce it, which is waste time and cost. Whereas, using biotechnology method to prepare collagen can reduce production cost. Prolyl4-hydroxylase, the key enzyme of collagen synthesis, which play an important role in sustaining collagen structure stability. So this experiment co-expression of type Ⅲ collagen and the key enzymes P4H to get the target protein which has application value.The studies are as follows: Using the optimized hydroxylase gene construct P4H (Prolyl4-hydroxylase) expression vector, then electroporating to X33, at last generating contains P4H P.pastoris recombinant X33/a PDI/a strains.Optimizing type Ⅲ collagen gene and constructing the expression vector, electroporate to recombinant strains contained P4H. Expression in the recombinant P. pastoris strains was induced with methanol, and the cells were harvested72h after induction and broken in a lysate buffer. Soluble fractions of cell lysates from strains were analyzed by8%SDS-PAGE under reducing conditions. And through the mass spectrometry to determine type III collagen molecule, which protein size is150kDa. Whereas, the protein size is120kDa after digested by pepsin.Fermentation condition was optimized in shake flask at first, the quantity of protein expression has improved after the high density fermentation. Trying to use different methods to purify collagen:salting out, molecular sieves, and ion exchange, finally found the salting out is a good method to get high purity of collagen.Identification the nature of protein. Firstly, using mass spectrometry analysis of collagen hydroxylation level; Secondly, compared with standard protein, analysis of the triple helical structure of collagen by CD; Thirdly, SDS-PAGE analysis under nonreducing conditions of disufide-bonded trimer formation of recombinant a1(Ⅲ) chains obtained by purified. Finally, the fibrils were analyzed using electron microscope. The above properties are consistent with standard type Ⅲ collagen’s characteristics.