SummaryFifteen related ligation-independent cloning vectors were constructed for high-throughput cloning and purification of proteins. The vectors encode a TEV protease site for removal of tags that facilitate protein purification (his-tag) or improve solubility (MBP, GST). Specialized vectors allow coexpression and copurification of interacting proteins, or in vivo removal of MBP by TVMV protease to improve screening and purification. All target genes and vectors are processed by the same protocols, which we describe here.

Production of milligram quantities of numerous proteins for structural and functional studies requires an efficient purification pipeline. We found that the dual tag, his 6 -tag-maltose-binding protein (MBP), intended to facilitate purification and enhance proteins' solubility, disrupted such a pipeline, requiring additional screening and purification steps. Not all proteins rendered soluble by fusion to MBP remained soluble after its proteolytic removal, and in those cases where the protein remained soluble, standard purification protocols failed to remove completely the stoichiometric amount of his 6 -tagged MBP generated by proteolysis. Both liabilities were alleviated by construction of a vector that produces fusion proteins in which MBP, the his 6 -tag and the target protein are separated by highly specific protease cleavage sites in the configuration MBP-site-his 6 -site-protein. In vivo cleavage at the first site by co-expressed protease generated untagged MBP and his 6 -tagged target protein.Proteins not truly rendered soluble by transient association with MBP precipitated, and untagged MBP was easily separated from the his-tagged target protein by conventional protocols. The second protease cleavage site allowed removal of the his 6 -tag. KeywordsHigh-throughput; Structural genomics; Maltose-binding protein; TVMV protease; Ligationindependent cloningThe burgeoning genomic information now available makes vast numbers of proteins accessible for structural and functional studies, and many large-scale projects have developed automated protocols for amplifying, cloning, and expressing genes, and for screening proteins for desirable properties [1][2][3][4][5]. Similar strides have been made in streamlining protein purification, but production of sufficient material for detailed structural and functional characterization remains labor-intensive and time-consuming [3,4,6,7]. Typically, purification is facilitated by fusing proteins to affinity tags, most commonly a his-tag, which allows purification by immobilized metal-ion affinity chromatography (IMAC,[8]). Additional tags are often attached to improve proteins' solubility, such as maltose-binding protein (MBP) [2][3][4]9,10]. In typical protein production pipelines, the resulting fusion proteins are first screened for ☆ This manuscript has been created by the University of Chicago as operator of Argonne National Laboratory under Contract No.W-31-109-ENG-38 with the US Department of Energy. The US government retains for itself, and others acting on its behalf, a paid-up, nonexclusive, irrevocable worldwide license in said article to reproduce, prepare derivative works, distribute copies to the public, and perform publicly and display publicly, by or on behalf of the government. The US Government's right to retain a nonexclusive royaltyfree license in and to the copyright covering this paper, for governmental purposes, is acknowledged. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript solubility, then purified by semi-robotic p...

Escherichia coli NZN111 is blocked in the ability to grow fermentatively on glucose but gave rise spontaneously to a mutant that had this ability. The mutant carries out a balanced fermentation of glucose to give approximately 1 mol of succinate, 0.5 mol of acetate, and 0.5 mol of ethanol per mol of glucose. The causative mutation was mapped to the ptsG gene, which encodes the membrane-bound, glucose-specific permease of the phosphotransferase system, protein EIICB glc . Replacement of the chromosomal ptsG gene with an insertionally inactivated form also restored growth on glucose and resulted in the same distribution of fermentation products. The physiological characteristics of the spontaneous and null mutants were consistent with loss of function of the ptsG gene product; the mutants possessed greatly reduced glucose phosphotransferase activity and lacked normal glucose repression. Introduction of the null mutant into strains not blocked in the ability to ferment glucose also increased succinate production in those strains. This phenomenon was widespread, occurring in different lineages of E. coli, including E. coli B.

Fermentative production of succinic acid from glucose by Escherichia coli was significantly increased by overexpression of phosphoenolpyruvate carboxylase. In contrast, overexpression of phosphoenolpyruvate carboxykinase had no effect. Under optimized conditions, induction of the carboxylase resulted in a 3.5-fold increase in the concentration of succinic acid, making succinic acid the major fermentation product by weight.

Energy-efficient capture of CO2 from power-plant flue gas is one of the grand challenges to reduce greenhouse gas (GHG) emissions. Current CO2-capture technologies are limited by parasitic energy loss, inefficient capture, and unfavorable process economics. We present a novel electrochemical method for CO2 capture from coal-fired power-plant flue gas. The method utilizes in-situ electrochemical pH control with a resin wafer electrodeionization (RW-EDI) device that continuously shifts the pH of the process fluid between basic and acidic in sequential chambers (pH swing). This pH swing enables capture of CO2 from flue gas in the basic chamber followed by release (recovery) of the captured CO2 (purified) in the acidic chamber of the same device. The approach is based on the sensitivity of the thermodynamic equilibrium of CO2 hydration/dehydration reactions over a narrow pH range. The method enables simultaneous absorption (capture) of CO2 from flue gas and desorption (release) at atmospheric pressure without heating, vacuum, or consumptive chemical usage. In other words, the method concentrates CO2 from ∼15% in flue gas to >98% in the recovery stream. To the best of our knowledge, this is the first experimental study focusing on simultaneous capture and release (recovery) of CO2 using an electrochemical method. We describe the method, the role of operating parameters on CO2 recovery, and advancements in process design and engineering for improved efficiency. We report on a method to enhance gas/liquid mixing inside the RW-EDI, which significantly increased CO2 capture rates. We also discuss the importance of using an enzyme/catalyst in enhancing the reaction kinetics. CO2 capture was observed to be a strong function of gas and liquid flow rates and applied electrical field. Up to 80% of the CO2 was captured from a simulated flue gas stream with >98% purity. The results indicate that a narrow pH swing from 8 to 6 (near-neutral pH) could offer a viable pathway for energy-efficient CO2 capture if the reaction kinetics are enhanced. Carbonic anhydrase enzyme enhances the reaction kinetics at near-neutral pH; however, the enzyme lost activity due to the instability at the operating conditions. This observation highlighted the necessity of robust enzymes/catalysts to enhance kinetics of CO2 recovery near-neutral pH.

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A simplified approach developed recently for the production of heterologous proteins in Escherichia coli uses 2-liter polyethylene terephthalate beverage bottles as disposable culture vessels [Sanville Millard, C. et al. 2003. Protein Expr. Purif. 29, 311-320]. The method greatly reduces the time and effort needed to produce native proteins for structural or functional studies. We now demonstrate that the approach is also well suited for production of proteins in defined media with incorporation of selenomethionine to facilitate structure determination by multiwavelength anomalous diffraction. Induction of a random set of Bacillus stearothermophilus target genes under the new protocols generated soluble selenomethionyl proteins in good yield. Several selenomethionyl proteins were purified in good yields and three were subjected to amino acid analysis. Incorporation of selenomethionine was determined to be greater than 95% in one protein and greater than 98% in the other two. In the preceding paper [Zhao et al., this issue, pp. 87-93], the approach is further extended to production of [U-15N]- or [U-13C, U-15N]-labeled proteins. The approach thus appears suitable for high-throughput production of proteins for structure determination by X-ray crystallography or nuclear magnetic resonance spectroscopy.

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