Recombinant Collagen Trimers from Insect Cells and Yeast - Methods in Molecular Biology: Extracellular Matrix Protocols - page 41

Fig. 1. SDS-PAGE analysis of pepsin digested recombinant human collagens
expressed in H5 insect cells. Long arrow indicates the 1 chains of type III (lane 1)

and type II collagens (lane 2), the type I collagen homotrimer (lane 3), and the type I
collagen heterotrimer (lane 4). Short arrow indicates the

α

α 2 chain of type I col-

lagen heterotrimer.

back to 7.4 and analyze the samples by SDS-PAGE ( Fig. 1 ). The thermal stabil-
ity of the pepsin digested recombinant human type III collagen can be studied by
trypsin-chymotrypsin digestion (19) . Preheat samples at the selected tempera-
tures for 5 min and digest for 2 min with a mixture of 100 µ g/mL trypsin and

250 g/mL chymotrypsin. Terminate the digestion by adding trypsin inhibitor to

a final concentration of 0.5 mg/mL and analyze the samples by SDS-PAGE.

µ

4. Expression of recombinant human type III procollagen in H5 cells can be scaled

up by using suspension cultures in shaker flasks or bioreactors. The largest
amount of recombinant type III procollagen we have obtained from H5 cells cul-
tured in shaker flasks has been about 60 mg/L ( see Note 8 ). The recombinant
type III collagen produced in H5 cells was found to be very similar in its 4-hydrox-
yproline content and T m to type III collagen extracted from various tissues,
whereas its hydroxylysine content was found to be about 60% of that of the non-
recombinant type III collagen (9) .

3.2. Expression of Recombinant Human Type III Procollagen
inPichia pastoris
3.2.1. Generation of Recombinant Pichia Expression Vectors
1.

Generate a modified Pichia expression vector pARG815 by replacing the HIS4
selection marker in pAO815 with the S. cerevisiae ARG4 selection marker. Digest
pYM25 containing the ARG4 gene with Hpa I, and ligate the ARG4 Hpa I frag-
ment into Eco RV-digested pAO815.