A less laborious approach to the high-throughput production of recombinant proteins in Escherichia coli using 2-liter plastic bottles.
Contemporary approaches to biology often call for the high-throughput production of large amounts of numerous proteins for structural or functional studies. Even with the highly efficient protein expression systems developed in Escherichia coli, production of these proteins is laborious and time-consuming. We have simplified established protocols by the use of disposable culture vessels: common 2-liter polyethylene terephthalate beverage bottles. The bottles are inexpensive, fit conveniently in commonly available flask holders, and, because they are notched, provide sufficient aeration to support the growth of high-density cultures. The use of antibiotics and freshly prepared media alleviates the need for sterilization of media and significantly reduces the labor involved. Uninoculated controls exhibited no growth during the time required for protein expression in experimental cultures. The yield, solubility, activity, and pattern of crystallization of proteins expressed in bottles were comparable to those obtained under conventional culture conditions. After use, the bottles are discarded, reducing the risk of cross-contamination of subsequent cultures. The approach appears to be suitable for high-throughput production of proteins for structural or functional studies.
Millard
Cynthia Sanville
CS
Environmental Research Division, Argonne National Laboratory, Bldg. 202/Rm. BE111, 9700 South Cass Avenue, Argonne, IL 60439, USA.
Stols
Lucy
L
Quartey
Pearl
P
Kim
Youngchang
Y
Dementieva
Irina
I
Donnelly
Mark I
MI
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019149:Bioreactors; D001709:Biotechnology / Q000295:instrumentation* / Q000379:methods*; D002478:Cells, Cultured; D003460:Crystallization; D003470:Culture Media; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism*; D015870:Gene Expression; D023281:Genomics / Q000379:methods; D007700:Kinetics; D011093:Polyethylene Terephthalates; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D012995:Solubility
D001426:Bacterial Proteins; D003470:Culture Media; D011093:Polyethylene Terephthalates; D011994:Recombinant Proteins