A comparison of the process issues in expressing the same recombinant enzyme periplasmically in Escherichia coli and extracellularly in Streptomyces lividans.
The choice of a host for the production of a biological molecule will have a significant effect on isolation and purification procedures employed. This paper makes a comparison between the production of a single enzyme, a recombinant alpha-amylase, in Escherichia coli and Streptomyces lividans, on a small scale. It defines the differences in the cultivation and in the isolation stages and also describes the impact of the expression system on later downstream processing steps. At the cultivation stage, the specific productivity of the E. coli in units per gram per hour is four times that of the S. lividans while the total biomass yields are of the same order. The initial volume for downstream processing of S. lividans is six-fold larger and the total protein released into the extracellular medium is three times greater than E. coli, however, the recoverable yield from the E. coli is a fifth of that obtained from the S. lividans and requires three additional stages prior to chromatography. Even with these stages the final specific activity is 64% of the S. lividans. The results indicate the need to consider the whole process when making such comparisons.
Pierce
J J
JJ
GlaxoWellcome, Langley Court, South Eden Park Road, Beckenham, Kent BR3 3BS, UK.
Robinson
S C
SC
Ward
J M
JM
Keshavarz-Moore
E
E
Dunnill
P
P
D018533:Biomass; D001709:Biotechnology; D002847:Chromatography, Agarose; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D005285:Fermentation; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D019897:Periplasm / Q000201:enzymology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D000516:alpha-Amylases / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification
D011994:Recombinant Proteins; D000516:alpha-Amylases