Protein production and purification

Gräslund, S.; Nordlund, P.; Weigelt, J.; Hållberg, Bengt; Bray, James E.; Gileadi, O.; Knapp, Stefan; Oppermann, U.; Arrowsmith, C.H.; Hui, Raymond; Ming, Jing; Dhe‐Paganon, Sirano; H-W., Park; Savchenko, Alexei; Yee, Adelinda; Edwards, A.M.; Vincentelli, Renaud; Cambillau, Christian; Kim, R; Sh, Kim; Rao, Zihe; Shi, Yunyu; Terwilliger, Thomas C.; Cy, Kim; Hung, Li Wei; Waldo, Geoffrey S.; Peleg, Yoav; Albeck, Shira; Unger, Tamar; Dym, Orly; Prilusky, Jaime; Sussman, Joel L.; Stevens, Raymond C.; Lesley, Scott A.; Wilson, Ian A.; Joachimiak, Andrzej; Collart, F.; Dementieva, I.; Donnelly, Mark I.; Eschenfeldt, William H.; Kim, Y; Stols, Lucy; Wu, R.; Zhou, Min; Burley, S.K.; Emtage, J.S.; Sauder, J.M.; Thompson, Devon A.; Bain, K.T.; Luz, José Maria Rodrigues da; Gheyi, T.; Zhang, F; Atwell, S.; Almo, Steven C.; Bonanno, Jeffery B.; Fiser, András; Swaminathan, Subramanyam; Studier, F. William; Chance, M. R. A.; Šali, Andrej; Acton, Thomas; Xiao, Rong; Zhao, Li; C, L; Hunt, J.F.; Tong, Liang; Cunningham, K.; Inouye, Masayori; Anderson, Stephen; Janjua, H.; Shastry, R.; Ho, C.K.; Wang, D; Wang, H; Jiang, Mei; Montelione, Gaetano T.; Stuart, David I.; Owens, Raymond J.; Daenke, Susan; Schütz, Anja; Heinemann, Udo; Yokoyama, Shigeyuki; Büssow, Konrad; Gunsalus, Kristin C.

doi:10.1038/nmeth.f.202

Cited by 746 publications

(320 citation statements)

References 89 publications

Supporting

Mentioning

317

Contrasting

Unclassified

Order By: Relevance

“…Site-directed mutagenesis was performed using Quikchange II site-directed mutagenesis kit (Stratagene). Proteins were produced using standard methods 28,29 , variations are described in the Supplementary Methods.…”

Section: Discussionmentioning

confidence: 99%

Lysine methylation of VCP by a member of a novel human protein methyltransferase family

et al. 2012

View full text Add to dashboard Cite

Valosin-containing protein (VCP, also called p97) is an essential and highly conserved adenosine triphosphate-dependent chaperone implicated in a wide range of cellular processes in eukaryotes, and mild VCP mutations can cause severe neurodegenerative disease. Here we show that mammalian VCP is trimethylated on Lys315 in a variety of cell lines and tissues, and that the previously uncharacterized protein mETTL21D (denoted here as VCP lysine methyltransferase, VCP-KmT) is the responsible enzyme. VCP methylation was abolished in three human VCPKmT knockout cell lines generated with zinc-finger nucleases. Interestingly, VCP-KmT was recently reported to promote tumour metastasis, and indeed, VCP-KmT-deficient cells displayed reduced growth rate, migration and invasive potential. Finally, we present data indicating that VCP-KmT, calmodulin-lysine methyltransferase and eight uncharacterized proteins together constitute a novel human protein methyltransferase family. The present work provides new insights on protein methylation and its links to human disease.

show abstract

Section: Discussionmentioning

confidence: 99%

Lysine methylation of VCP by a member of a novel human protein methyltransferase family

et al. 2012

View full text Add to dashboard Cite

show abstract

“…6 In the most common approaches to target validation, the scale of production and stringency of the selection criteria increase in a stepwise manner [ Fig. 1(b)].…”

Section: Resultsmentioning

confidence: 99%

“…[2][3][4][5] To meet their annual production goals, thousands of proteins or domains must be screened, 6 and this demands that traditional methods for molecular cloning and protein purification be adapted to parallel operation. 7 Automated processing of nucleic acids is now routinely performed using commercial instruments, but parallelized robotic protein purification has typically been achieved with costly customized systems 8 or at production scales that are insufficient for screening by NMR spectroscopy.…”

mentioning

confidence: 99%

Rapid, robotic, small‐scale protein production for NMR screening and structure determination

Jensen

Woytovich

et al. 2010

Protein Science

View full text Add to dashboard Cite

Three-dimensional protein structure determination is a costly process due in part to the low success rate within groups of potential targets. Conventional validation methods eliminate the vast majority of proteins from further consideration through a time-consuming succession of screens for expression, solubility, purification, and folding. False negatives at each stage incur unwarranted reductions in the overall success rate. We developed a semi-automated protocol for isotopically-labeled protein production using the Maxwell-16, a commercially available bench top robot, that allows for single-step target screening by 2D NMR. In the span of a week, one person can express, purify, and screen 48 different 15 N-labeled proteins, accelerating the validation process by more than 10-fold. The yield from a single channel of the Maxwell-16 is sufficient for acquisition of a high-quality 2D 1 H-15 N-HSQC spectrum using a 3-mm sample cell and 5-mm cryogenic NMR probe. Maxwell-16 screening of a control group of proteins reproduced previous validation results from conventional small-scale expression screening and large-scale production approaches currently employed by our structural genomics pipeline. Analysis of 18 new protein constructs identified two potential structure targets that included the second PDZ domain of human Par-3. To further demonstrate the broad utility of this production strategy, we solved the PDZ2 NMR structure using [U-15 N, 13 C] protein prepared using the Maxwell-16. This novel semi-automated protein production protocol reduces the time and cost associated with NMR structure determination by eliminating unnecessary screening and scale-up steps.

show abstract

“…265 code optimized synthetic genes were cloned into the NdeI site behind the RBS site, and 240 genes were over-expressed, which reached 85% success rate of enzyme expression. This is over two-fold as high as data collected from expression of 58 806 bacteria proteins previously [1]. A medium consisting of crude glycerol and corn steep liquor was optimized for culture of E. coli B, which increased the weight of harvested cells up to 50 g/L within 24 h. The low-cost medium and high-celldensity fermentation reduced the total cost of enzyme production by two thirds and was less susceptible to contamination by bacteria or phages.…”

mentioning

confidence: 91%

Tailor‐made biocatalysts enzymes for the fine chemical industry in China

Jiang

Tao

Yang

2016

Biotechnology Journal

View full text Add to dashboard Cite

The Center of Industrial Biotechnology (CIBT) was established in Huzhou for fine chemicals in 2006 and CIBT Shanghai was founded for bulk chemicals in 2008. CIBT is a non‐profit organization under auspices of the Shanghai Institutes for Biological Sciences, Shanghai Branch of the Chinese Academy of Sciences (CAS) and Huzhou Municipal Government. CIBT is affiliated with the CAS, which enables it to take advantage of the rich R&D resources and support from CAS; yet CIBT operates as an independent legal entity. The goal of CIBT is to incubate industrial biotechnologies and accelerate the commercialization of these technologies with corporate partners in China.

show abstract

Protein production and purification

Cited by 746 publications

References 89 publications

Lysine methylation of VCP by a member of a novel human protein methyltransferase family

Lysine methylation of VCP by a member of a novel human protein methyltransferase family

Rapid, robotic, small‐scale protein production for NMR screening and structure determination

Tailor‐made biocatalysts enzymes for the fine chemical industry in China

Contact Info

Product

Resources

About