Recombinant Collagen Trimers from Insect Cells and Yeast - Methods in Molecular Biology: Extracellular Matrix Protocols - page 38

pastoris . These methods can be applied to the expression of other recombinant
collagen types, both homotrimeric and heterotrimeric (10 , 11) .

2. Materials
2.1. Materials for Expression
of Recombinant Human Type III Procollagen in Insect Cells

1. pVL1392 and p2Bac baculovirus expression vectors (Invitrogen, San Diego, CA).

2. Full-length cDNAs for the α - (12) and β - (13) subunits of human prolyl 4-hydroxy-

1(III)] (14) .
3. Wizard Plus Maxiprep DNA purification system (Promega, Madison, WI).
4. Sf9 and High Five (H5) insect cells (Invitrogen).
5. TNM-FH medium (Sigma, St. Louis, MO) supplemented with 10% fetal bovine
serum (FBS).
6. Six-well plates and 60- and 100-mm tissue-culture Petri dishes.
7. BaculoGold DNA and Transfection Buffers A: Grace's Medium with 10% FBS
and B: 25 m M HEPES, pH 7.1, 125 m M CaCl 2 , 140 m M NaCl (Pharmingen, San
Diego, CA).
8. 3% seaplaque low-melting-point agarose (FMC, Rockland, ME) in H 2 O (autoclaved).
9. L-ascorbic acid phosphate (Wako, Osaka, Japan).
10.

lase and the pro α 1 chain of human type III procollagen [pro α

PBS: 0.15 M NaCl, 20 m M phosphate, pH 7.4.

11. Homogenization buffer (300 m M NaCl, 0.2% Triton X-100, and 70 m M Tris,

pH 7.4).

12. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) apparatus.

13. PIIINP radioimmunoassay (Farmos Diagnostica, Turku, Finland).

14. Pepsin (Boehringer Mannheim, Mannheim, Germany), trypsin type XIII (Sigma),

chymotrypsin type VII (Sigma), trypsin inhibitor type II-S (Sigma).

2.2. Materials for Expression
of Recombinant Human Type III Procollagen
in the Yeast Pichia pastoris

1. pAO815, pPIC9, and pPICZ B Pichia pastoris expression vectors (Invitrogen).

pYM25 containing the Saccharomyces cerevisiae ARG4 selection marker
(obtained from Dr. James Cregg, Keck Graduate Institute of Applied Life
Sciences, Claremont, CA.

2. his4, arg4 Pichia pastoris host strain (obtained from Dr. James Cregg).

3. Yeast extract peptone dextrose medium (YPD: 1% yeast extract, 2% peptone, 2%

dextrose), minimal dextrose medium (MD: 1.34% yeast nitrogen base with
ammonium sulfate and without amino acids [YNB], 4 10 -5 % biotin, 1% dex-

trose), buffered glycerol-complex medium (BMGY: 0.1% yeast extract, 0.2%
peptone, 100 m M potassium phosphate, pH 6.0, 1.34% YNB, 4

×

10 -5 % biotin,
1% glycerol), buffered minimal methanol medium (BMM: 100 m M potassium
phosphate, pH 6.0, 1.34% YNB, 4

×

× 10 -5 % biotin, 0.5% methanol). Recipes for