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High Yield Expression of Proteins in E. Coli for NMR Studies Advances in Bioscience and Biotechnology, 2013, 4, 751-767 ABB http://dx.doi.org/10.4236/abb.2013.46099 Published Online June 2013 (http://www.scirp.org/journal/abb/) High yield expression of proteins in E. coli for NMR studies Somnath Mondal1,2, Divya Shet1, Chinmayi Prasanna 1, Hanudatta S. Atreya1,2* 1NMR Research Centre, Indian Institute of Science, Bangalore, India 2Solid State and Structural Chemistry Unit, Indian Institute of Science, Bangalore, India Email: *[email protected] Received 1 April 2013; revised 12 May 2013; accepted 25 May 2013 Copyright © 2013 Somnath Mondal et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT increase in size of the molecules the sensitivity of NMR experiments decrease and hence isotopic enrichment helps In recent years, high yield expression of proteins in E. to alleviate to some extent the deleterious effects of large coli has witnessed rapid progress with developments size. of new methodologies and technologies. An important Isotope labeling implies the replacement of 12C, 14N or advancement has been the development of novel re- 1H atoms of the backbone or side-chain of proteins by combinant cloning approaches and protocols to ex- 13C, 15N or 2H, respectively, either uniformly through- press heterologous proteins for Nuclear Magnetic Re- out the protein (i.e., independent of the amino acid type) sonance (NMR) studies and for isotopic enrichment. or in a selective manner (i.e., amino acid type dependent) Isotope labeling in NMR is necessary for rapid acqui- [2]. This can be accomplished in one of the three ways: 1) sition of high dimensional spectra for structural stu- over-expression of proteins in E. coli or higher organ- dies. In addition, higher yield of proteins using vari- isms if required; 2) use of cell-free protein expression; ous solubility and affinity tags has made protein over- and 3) chemical synthesis. Of the three, over-expression expression cost-effective. Taken together, these me- of proteins in E. coli is by far the most popular and cost- thods have opened new avenues for structural stu- effective method today. The protein to be expressed is dies of proteins and their interactions. This article first cloned using the recombinant DNA methods. The deals with the different techniques that are employed protein is either expressed as such or fused with a suit- for over-expression of proteins in E. coli and different able tag for increasing the yield/solubility and/or facili- methods used for isotope labeling of proteins vis-à-vis tating easy purification. Starting from 1960s when the NMR spectroscopy. first isotope enrichment of proteins was carried out, iso- tope labeling has come a long way with various advan- Keywords: E. Coli; Recombinant DNA Technology; cements. Today it is possible to express proteins with 13C Structural Biology; NMR Spectroscopy 15 and/or N at a fraction of cost and/or with an order of magnitude compared to a decade ago. 1. INTRODUCTION In this article, we focus on the different techniques used today for over-expression and isotope labeling of The two most popular methods today for structural stu- proteins in E. coli for NMR studies. The various methods dies of biomolecular systems are X-ray crystallography proposed recently for high-yield expression are discussed and Nuclear Magnetic Resonance (NMR) spectroscopy. with suitable examples. The article is divided into three The former requires the preparation of single crystal of sections. In the first section, the different general proto- proteins whereas the latter involves the preparation of cols for protein expression are described. In the second protein samples either in solution (for liquid state NMR) section, different methods for isotope labeling for NMR or in micro-crystalline form (for solid state NMR). In the studies are discussed. In the last section, special proto- case of NMR studies of proteins, homonuclear one- and cols for expressing difficult proteins in E. coli for NMR two-dimensional (1D/2D) techniques are sufficient if the are covered. molecular mass of the system is less than 10 kDa (<90 - 100 amino acid residues) [1]. However, for larger pro- teins advanced NMR techniques combined with isotopic 2. RECENT METHODS FOR HIGH enrichment or isotope labeling of the molecule (with 13C/ YIELD PROTEIN EXPRESSION IN 15N/2H) is required [2]. This is owing to the fact that with E. COLI *Corresponding author. Purification of proteins in Escherichia coli at a very high OPEN ACCESS 752 S. Mondal et al. / Advances in Bioscience and Biotechnology 4 (2013) 751-767 yield requires over production by cells. A major impede- is transferred into same volume of minimal media when ment in this regard is that many proteins are poorly ex- the cells are in the middle of growing phase. Further, the pressed especially those of eukaryotic origin, and some- cells are cultured for medium exchange at pre-deter- times in insoluble form which are prone to degradation mined, optimized temperature for the target protein pro- by cellular proteases. In recent years, high level expres- duction [7]. At this point, the bacterial culture is induced sion of proteins has been impelled by various develop- for target protein production by IPTG at the same tem- ments in understanding and manipulating the biological perature for an optimized time period. Bacterial expres- processes of E. coli. sion parameters to be optimized in this method to attain a Paramount factors to obtain high yields of protein are very high cell density in laboratory shaking cultures are: gene of interest, expression vector, gene dosage, tran- 1) double selection of colonies highly expressing the scriptional regulation, codon usage, translational regula- target protein; 2) optimization of temperature and time tion, host design, growth media and culture conditions or period for starter culture to avoid plasmid instability or fermentation conditions available for manipulating the loss; 3) optimizing induction temperature and time cour- expression conditions, specific activity or biological ac- se; and 4) glucose optimization. These expression para- tivity of the protein of interest, protein targeting, fusion meters must be optimized for every new target protein proteins, molecular chaperones, protein degradation [3-6]. [7]. Recent methods to obtain high protein expression include High cell density shaking cultures by auto-induction is high cell density shaking cultures, High cell density fer- an efficient cost effective method for high level protein mentation, fed batch based cultivation, cold shock induc- production with glycerol as carbon source since glucose tion, co-expression with chaperones or fusion tags, cell prevents auto-induction and lactose is necessary for auto- free protein synthesis among others. Figure 1 below de- induction at previously optimized physiological condi- picts some of the methods used to achieve high yielding tions and optimized minimal medium [4,9]. Auto-indu- proteins. Each of these methods is described below in ction method can be alternative to tedious fermenter cul- detail. tures as the latter requires additional equipment to moni- tor various parameters which is economically prohibitive. 2.1. High Cell Density Shaking Cultures Additionally, the smaller media volumes required make the production method ideal for selective side-chain or High cell density shaking cultures based on IPTG induct- amino acid labeling procedures [10,11]. Auto-induction ion reported by Qianqian Li and others used a regular medium contains NH4 as nitrogen source, glycerol, incubator shaker to achieve a cell-density (measured as lactose and glucose at optimized levels so that glycerol is optical density at 600 nm; OD 600) of 10 - 20 in the nor- used as carbon source. Lactose is metabolized for auto- mal laboratory setting instead of a fermenter [7]. Several induction once glucose is depleted, which is otherwise parameters such as host strain selection, plasmid copy the most favorable carbon source. Individual isotopes numbers, promoter selection, mRNA stability, and codon (13C/15N) incorporation is slightly more efficient than 15N usage were optimized to attain a high cell density [3,7,8]. and 13C incorporated together. To overcome decreased In this IPTG induction based method, rich media is used rate of incorporation of appropriate isotopes, a longer as starter culture, which is grown at an optimized tem- period of clearance of unlabeled nutrients is allotted be- perature and time period, following which the cell pellet fore induction. Also, it favors metabolic rates of E. coli in deuterated media [11] and high yield expression. Gen- erally a lower temperature for prolonged duration of time is preferred to obtain soluble target protein at high yield without any additional requirement of amino acids and vitamin supplements [9]. 2.2. Fed Batch Cultivation Method This refers to a fed batch liquid phase cultivation tech- nology using enzymatic release of glucose for protein production in E. Coli in round-bottom Erlenmeyer sha- ken cultures [12]. Media comprising mixture of mineral salts and complex additives is optimized to provide high cell density and high protein yield. High cell density and favorable physiological conditions of the target protein ® Figure 1. Depiction of different methods leading to high yield are achieved in the shaken cultures with EnBase Flo protein production in Escherichia coli. (glucose releasing polymer is in soluble form rather than Copyright © 2013 SciRes. OPEN ACCESS S. Mondal et al. / Advances in Bioscience and Biotechnology 4 (2013) 751-767 753 a gel) throughout the protein expression period which is temperature for prolonged hours. Thus obtained crude a day longer compared to other expression methods but cell lysate is amenable for NMR studies making this a reaches OD600 of 30 to 50 [12] yielding high amount of rapid high yield protein production method [16]. En- soluble protein in comparison to commonly used media.
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