Recombinant Collagen Trimers from Insect Cells and Yeast - Methods in Molecular Biology: Extracellular Matrix Protocols - page 44

the expression vectors must therefore include at least 1.7 kbp of baculovirus DNA
downstream of the polyhedrin stop codon to counteract the lethal deletion. The
recombination frequency with BaculoGold DNA is at least 99%, which is a major
improvement over the 0.1% recombination frequency obtained with the
wild-type baculovirus DNA.

3. In spite of the high recombination frequency with the BaculoGold DNA and the

fact that wild-type baculovirus DNA expressing polyhedrin cannot be formed
during recombination, it is still advisable to plaque-purify the recombinant virus
stocks, as aberrant crossing over during transfection can lead to total or partial
deletions of the recombinant gene of interest. The resultant virus stock after
plaque-purification is derived from a single virus clone.

4. If plaques are not clearly visible, the plaque assay plates can be stained with

Neutral Red or MTT (thiazolyl blue), for example (21) .

5. The MOI (multiplicity of infection = PFU/cell number) should be below 1 during

all amplifications, as values greater than 1 can lead to deletions in the recombi-
nant virus particles (20) . The PFU/mL of a virus stock can be obtained from a
plaque assay by the formula: PFU/mL = 1/virus dilution (e.g., 10 -5 ) × number of

plaques × 1/mL virus dilution added to the plate.

6. H5 cells consistently give 3- to 10-fold higher expression levels than Sf9 cells for

recombinant human type III procollagen (9) .

7. Most of the recombinant fibril-forming human type I, II, or III procollagens pro-

duced (70-90%) is retained within the insect cells (9-11) .

8. The highest expression levels obtained for type I and II collagens in suspension

culture have been 20-40 mg/L and 50 mg/L, respectively (10 , 11) .

9. The signal sequence of the prolyl 4-hydroxylase subunit was found to play a

major role in enzyme assembly in Pichia (8) . The authentic human signal
sequence was ineffective for transport of the

β

-subunit into the lumen of the
endoplasmic reticulum, as only trace amounts of an active

β

α 2 β 2 enzyme tetramer
were produced. The highest tetramer assembly level was obtained with the
S. cerevisiae mating factor pre-pro sequence, whereas that obtained with the

P. pastoris acid phosphatase 1 signal sequence was about 40% of this value.

α

10. The doubling time of the his4, arg4 P. pastoris strain is approximately 2 h in YPD.

11. P. pastoris cells can be pulsed using the parameters suggested for S. cerevisiae

by the manufacturer of the electroporation device.

12. Zeocin is active only at a low salt concentration (<90 m M ) and at pH 7.5. There-

fore, screening for His + , Arg + phenotype and zeocin resistance must take place in
parallel plates, as the pH of MD plates is not suitable for zeocin selection.

13. As in the case of insect cells ( see Note 7 ), the vast majority of the recombinant

human type III procollagen in the Pichia expression system, too, is retained within
the cells and can thus be obtained from the soluble fraction of the cell lysates.

References
1. Prockop, D. J. and Kivirikko, K. I. (1995) Collagens: Molecular biology, dis-
eases, and potentials for therapy. Annu. Rev. Biochem. 64, 403-434.