Abstract
As the natural extension of the genomic sequencing projects, the goal of the various world-wide Structural Genomics projects is development of techniques for high throughput (HTP) cloning, protein overexpression, purification and structural determination, with the ultimate goal of determining all possible protein structures. Rapid (small-scale) screening of potential expression clones under different growth conditions is presumed to be possible and a viable way to increase throughput of protein expression. In order to test the utility of screening for soluble, heterologous protein expression, we have compared the production of recombinant proteins on a small scale (1 ml cultures in 96-well plates) in Escherichia coli under two growth conditions [a rich medium and a defined (minimal) medium] using an enzyme-linked immunosorbent assay (ELISA) against the affinity tag, with the amount of recombinant protein produced during the large-scale (500 ml) growth of E. coli. The large-scale expression products were examined after a single step affinity purification by visualization on SDS-PAGE gels. Of the open reading frames that were successfully expressed on the 1 ml scale as judged by immunodetection, 80% of them successfully scaled-up to 500 ml in a rich medium and 81% of them scaled-up in a defined medium. This is significantly higher than would be expected by a randomly selected expression condition and validates the use of small-scale expression as a screening tool for more efficient protein production.
Similar content being viewed by others
Abbreviations
- BSA:
-
bovine serum albumin
- c f :
-
final concentration
- ELISA:
-
enzyme-linked immunosorbent assay
- HTP:
-
high throughput
- IMAC:
-
immobilized metal affinity chromatography
- IPTG:
-
isopropyl-β-D-thiogalactopyranoside
- LSE:
-
large-scale expression
- NMR:
-
nuclear magnetic resonance
- ORF:
-
open reading frame
- PBS:
-
phosphate-buffered saline
- PCR:
-
polymerase chain reaction
- PMSF:
-
phenylmethanesulfonyl fluoride
- pNPP:
-
p-nitrophenyl phosphate
- SDS-PAGE:
-
sodium dodecyl sulfate polyacrylamide gel electrophoresis
- SSE:
-
small-scale expression
References
M.W.W. Adams H.A. Dailey L.J. Delucas M. Luo J.H. Prestegard J.P. Rose B.C. Wang (2003) Acc. Chem. Res. 36 191–198 Occurrence Handle10.1021/ar0101382 Occurrence Handle12641476
G. Fiala K.O. Stetter (1986) Arch. Microbiol. 145 56–61 Occurrence Handle10.1007/BF00413027
F.T. Robb D.L. Maeder J.R. Brown J. DiRuggiero M.D. Stump R.K. Yeh R.B. Weiss D.M. Dunn (2001) Methods Enzymol. 330 134–157 Occurrence Handle11210495
Weiss, R.B., Dunn, D.M., Robb, F.T. and Brown, J.R. (2003) NCBI GenBank direct submission
S.C. Makrides (1996) Microbiol. Rev. 60 512–538 Occurrence Handle8840785
G. Hannig S.C. Makrides (1998) TIBTECH 16 54–60
F.A.O. Marston (1986) Biochem. J. 240 1–12 Occurrence Handle3548705
H. Lilie E. Schwarz R. Rudolph (1998) Curr. Opin. Biotech. 9 497–501 Occurrence Handle10.1016/S0958-1669(98)80035-9 Occurrence Handle9821278
K-J. Kim H.E. Kim K-H. Lee W. Han M-J. Yi J. Jeong B-H. Oh (2004) Protein Sci. 13 1698–1703 Occurrence Handle10.1110/ps.04644504 Occurrence Handle15133160
R.K.C. Knaust P. Nordlund (2001) Anal. Biochem. 297 79–85 Occurrence Handle10.1006/abio.2001.5331 Occurrence Handle11567530
M. Boettner B. Prinz C. Holz U. Stahl C. Lang (2002) J. Biotechnol. 99 51–62 Occurrence Handle10.1016/S0168-1656(02)00157-8 Occurrence Handle12204557
S.P. Chambers D.A. Austen J.R. Fulghum W.M. Kim (2004) Protein Expr. Purif. 36 40–47 Occurrence Handle10.1016/j.pep.2004.03.003 Occurrence Handle15177282
H. Nguyen B. Martinez N. Oganesyan R. Kim (2004) J. Struct. Funct. Genomics 5 23–27 Occurrence Handle10.1023/B:JSFG.0000029195.73810.86 Occurrence Handle15263840
L. Dieckman M. Gu L. Stols M.I. Donnelly F.R. Collart (2002) Protein Expr. Purif. 25 1–7 Occurrence Handle10.1006/prep.2001.1602 Occurrence Handle12071692
Y.-P. Shih W.-M. Kung J.-C. Chen C.-H. Yeh A.H.-J. Wang T.-F. Wang (2002) Protein Sci. 11 1714–1719 Occurrence Handle10.1110/ps.0205202 Occurrence Handle12070324
G.E. Folkers B.N.M. Buuren Particlevan R. Kaptein (2004) J. Struct. Funct. Genomics 5 119–131 Occurrence Handle10.1023/B:JSFG.0000029200.66197.0c Occurrence Handle15263851
J. Sambrook D.W. Russell (2001) Molecular Cloning: A Laboratory Manual EditionNumber3 Cold Spring Harbor Laboratory Press Cold Spring Harbor, NY
M.M. Carrió A. Villaverde (2002) J. Biotechnol. 96 3–12 Occurrence Handle10.1016/S0168-1656(02)00032-9 Occurrence Handle12142138
A.P.J. Middelberg (2002) TRENDS Biotechnol. 20 437–443 Occurrence Handle10.1016/S0167-7799(02)02047-4 Occurrence Handle12220907
D. Kihara T. Shimizu M. Kanehisa (1998) Protein Eng. 11 961–970 Occurrence Handle10.1093/protein/11.11.961 Occurrence Handle9876916
C. Pasquier S.J. Hamodrakas (1999) Protein Eng. 12 631–634 Occurrence Handle10.1093/protein/12.8.631 Occurrence Handle10469822
M. Gomi F. Akazawa S. Mitaku (2000) Genome Inform. 11 414–415
B. Miroux J.E. Walker (1996) J. Mol. Biol. 260 289–298 Occurrence Handle10.1006/jmbi.1996.0399 Occurrence Handle8757792
G.J.S. Fowler A.T. Gardiner R.C. Mackenzie S.J. Barratt A.E. Simmons W.H.J. Westerhuis R.J. Cogdell C.N. Hunter (1995) J. Biol. Chem. 270 23875–23882 Occurrence Handle10.1074/jbc.270.40.23875 Occurrence Handle7559566
E. Kopetzki G. Schmacher P. Buckel (1989) Mol. Gen. Genet. 216 149–155 Occurrence Handle10.1007/BF00332244 Occurrence Handle2659969
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Sugar, F.J., Jenney, F.E., Poole, F.L. et al. Comparison of Small- and Large-scale Expression of Selected Pyrococcus furiosus Genes as an Aid to High-throughput Protein Production. J Struct Funct Genomics 6, 149–158 (2005). https://doi.org/10.1007/s10969-005-3341-3
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/s10969-005-3341-3