Production of mouse epidermal growth factor in yeast: high-level secretion using Pichia pastoris strains containing multiple gene copies.
We have constructed a synthetic secretion cassette encoding the alpha-factor prepro leader peptide from Saccharomyces cerevisiae fused to mouse epidermal growth factor (mEGF). This was used to compare the secretion of mEGF, a 53-amino acid polypeptide, in S. cerevisiae and Pichia pastoris. In both yeasts the leader sequence was accurately and efficiently cleaved showing that the S. cerevisiae-derived alpha-factor prepro region is correctly recognised and processed in P. pastoris. Of the total mEGF produced, over 90% was exported to the culture supernatant, although the final level of accumulation was dependent on the composition of the growth medium. With P. pastoris there was instability of the protein in minimal medium (yeast nitrogen base), probably caused by extracellular proteases. This was overcome by adding 1% Casamino acids and buffering the medium to pH 6.0. To increase the level of secreted mEGF we have developed a method for rapidly screening large numbers of P. pastoris transformants for the presence of many copies of a foreign gene. Using this procedure we isolated a strain containing 19 integrated copies of the mEGF gene which secreted 450 micrograms/ml of mEGF in high-density fermentations. Characterisation of the yeast-derived mEGF showed the presence of truncated forms, mEGF1-51 and mEGF1-52, as was found with S. cerevisiae-secreted human EGF [George-Nascimento et al., Biochemistry 27 (1988) 797-802]. In addition, the full-length protein, mEGF1-53, was secreted by P. pastoris.
Clare
J J
JJ
Wellcome Research Laboratories, Beckenham, Kent, U.K.
Romanos
M A
MA
Rayment
F B
FB
Rowedder
J E
JE
Smith
M A
MA
Payne
M M
MM
Sreekrishna
K
K
Henwood
C A
CA
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004815:Epidermal Growth Factor / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics; D007700:Kinetics; D051379:Mice; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011498:Protein Precursors / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D014170:Transformation, Genetic
D000596:Amino Acids; D005656:Fungal Proteins; C048632:MF(ALPHA)1 protein, S cerevisiae; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D004815:Epidermal Growth Factor