Comparisons of recombinant protein expression in diverse natural isolates of Escherichia coli.
We assessed heterologous protein expression in 64 strains obtained from the Escherichia coli Reference (ECOR) collection, a collection representing diverse natural E. coli populations. A plasmid generating a glutathione S-transferase and plant carbonic anhydrase fusion protein (GST-CA) under the control of the tac promoter was introduced into the ECOR strains, and the quantity of the fusion protein was determined by SDS-PAGE. The foreign protein was generated at various levels, from very high (40 strains, high producers) to very low (six strains, low producers). Immunoblotting showed that the high producers expressed approximately 250-500 times more GST-CA protein than the low producers. The results of semi-quantitative RT-PCR showed that the low producers generated mRNA levels comparable to those of the high producers, thereby suggesting that, at least in this case, inefficient translation is a major cause of the low production. We introduced a different plasmid, which expressed a maltose binding protein and plant guanylate kinase fusion protein (MBP-GK) into the six low producers. Interestingly, five of these expressed MBP-GK at very high levels. Thus, we conclude that the production of a particular protein from an expression vector can vary considerably, depending on the host strain. Strains in the ECOR collection could function as useful alternative hosts when a desired level of protein expression is not obtained from commonly used strains, such as E. coli K12 or B derivatives.
Jung
Yuna
Y
Department of Bioinformatics and Life Science, College of Natural Sciences, Soongsil University, Seoul 156-743, Korea.
Lim
Dongbin
D
D015153:Blotting, Western; D002256:Carbonic Anhydrases / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics; D004926:Escherichia coli / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics; D051528:Guanylate Kinases / Q000235:genetics; D058619:Maltose-Binding Proteins; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics
D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D051528:Guanylate Kinases; D002256:Carbonic Anhydrases