Recombinant Collagen Trimers from Insect Cells and Yeast - Methods in Molecular Biology: Extracellular Matrix Protocols - page 39

various Pichia media can be found in Version 3.0 of the Invitrogen Pichia
Expression Kit Manual (15) .

4. Zeocin (Invitrogen).

5. Triple-baffled Erlenmeyer flasks and silicone sponge closures (Sigma).

6. Electroporation device and cuvets.

7. Acid-washed glass beads (Sigma).

8. 1 M sorbitol.

9. 5% glycerol, 50 m M sodium phosphate buffer, pH 7.4.

3. Methods
3.1. Expression of Recombinant Human Type III Procollagen
in Insect Cells
3.1.1. Generation of Recombinant Baculovirus Expression Vectors
1.

Generate a double promoter baculovirus expression vector for recombinant
human prolyl 4-hydroxylase (p2Bac4PH αβ ) by cloning the cDNAs for its α - and

-subunits into the Not I site downstream of the p10 promoter and the Bam HI site
downstream of the polyhedrin promoter of p2Bac, respectively. Generate a Not I
site in the

β

-subunit cDNA 46 bp upstream of the translation initiation (ATG)
codon by PCR ( see Note 1 ).

α

2. Create a Bgl II site 16 bp upstream of the translation initiation codon to the full-

length cDNA for the pro 1 chain of human type III procollagen ( see Note 1 ).

Digest the cDNA with Bgl II and Xba I and ligate the insert to Bgl II- Xba I-digested
pVL1392, generating pVLrhproCIII.

α

3. Purify the recombinant expression vectors p2Bac4PH and pVLrhproCIII using

the Wizard Plus Maxiprep DNA purification system and sterilize them with a
0.22-

αβ

µ m syringe filter.

3.1.2. Generation of Recombinant Baculoviruses
1.

Generate the recombinant baculoviruses 4PH and rhproCIII by cotransfection

with BaculoGold baculovirus DNA ( see Note 2 ). Seed 2

αβ

10 6 Sf9 cells on 60- mm
tissue-culture Petri dishes. Allow cells to attach for at least 30 min. Remove the
culture medium from the plates and add 1 mL of Transfection Buffer A. Mix
BaculoGold DNA, 0.5

×

g of the baculovirus expression vector DNA
and incubate for 5 min at room temperature. Mix 1 mL of Transfection
Buffer B well with the DNA mixture, and add dropwise to the 60-mm insect cell
plate. Gently rock the plate after the addition of every 3-5 drops of the transfection
solution.
2. Incubate the plates at 27

µ g, with 5 µ

C for 4 h, remove the transfection solutions, and add
4 mL of TNM-FH medium supplemented with 10% FBS.

°

3. Incubate the plates at 27 C for 4 d. Collect the medium containing the recombi-

nant virus and amplify once by infecting 6

°

10 6 Sf9 cells in a 100-mm Petri dish

×

with 500 µ L of the collected transfection medium. Incubate the plates at 27 ° C for

3 d before harvesting the amplification medium.