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In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators.

The major cold shock protein of Eschenchia coli, CspA, produced upon a rapid downshift in growth temperature, Is involved in the transcriptional regulation of at least two genes. The protein shares high homology with the nucleic acid-binding do of the Y-box factors, a family of eukaryotic proteins involved in nscriptional and trandational regulation. The crystal structure of CspA has been determined at 2-A resolution and refined to R = 0.187. CspA Is composed of five antiparallel -strands forming a osed five-stranded (-barrel. The three-dimensional structure of CspA Is similar to that of the major cold shock protein of BaciUlus subtils, CspB, which has recently been determined at 2.45-A resolution. However, in contrast to CspB, no dimer Is formed in the crystal. The surface of CspA is characteristic for a protein interacting with single-stranded nucleic acds. Due to the high homology of the bacterial cold shock proteins with the Y-box factors, E. coft CspA and B. subdis CspB define a sructural framework for the common cold shock domain.The cold shock response inEscherichia colifollows an abrupt shift in growth temperature from 370C to 100C, inducing a lag phase in cell growth of 4-5 hr. It is accompanied by a severe reduction in protein synthesis (1). As a consequence of this cold shock, the relative rate of production of at least 14 cold shock proteins is increased. For 13 out of the 14 proteins the increase is 2-to 10-fold whereas synthesis of the major cold shock protein, CspA (CS 7.4), increases at least 100-fold, reaching a level of >10% oftotal protein synthesis at 100C (2). Other proteins expressed as part of the cold shock response of E. coli include NusA, RecA, polynucleotide phosphorylase, translation initiation factors 2a and 2p, pyruvate dehydrogenase (lipoamide), dihydrolipoamide acetyltransferase of pyruvate dehydrogenase, the nucleoid protein H-NS, and subunit A of DNA gyrase (1,3,4).CspA is a small hydrophilic protein consisting of 70 amino acids. It has striking similarity, at the level of 43% sequence identity (Fig. 1), with one domain ofthe Y-box factors, which is referred to as the cold shock domain (5, 6). The Y-box factors are a family of eukaryotic nucleic acid-binding proteins that preferentially bind to the Y box, an element of sequence CTAAIT-ClQYYAA found in the promoter regions of mammalian major histocompatibility complex class II genes (6). Within this sequence the underlined pentamer is especially conserved. Members of this family have also been found to bind to mRNA and to regulate translation in germ cells (7,8).CspA was shown to act as a transcriptional activator of the hns and gyrA genes encoding two other cold shock proteins (3,4). The promoter of hns contains one ATTGG element, whereas the promoter of gyrA contains three such elements, one of which is required for specific CspA-DNA interaction (4). ATTGG elements have been identified also in the promoter regions of genes encoding RecA, NusA, and polynucleotide phosphorylase, suggesting a common mechanism for induction...

The cold-shock response in both Escherichia coli and Bacillus subtilis is induced by an abrupt downshift in growth temperature. It leads to the increased production of the major cold-shock proteins, CS7.4 and CspB, respectively. CS7.4 is a transcriptional activator of two genes. CS7.4 and CspB share 43 per cent sequence identity with the nucleic acid-binding domain of the eukaryotic gene-regulatory Y-box factors. This cold-shock domain is conserved from bacteria to man and contains the RNA-binding RNP1 sequence motif. As a prototype of the cold-shock domain, the structure of CspB has been determined here from two crystal forms. In both, CspB is present as an antiparallel five-stranded beta-barrel. Three consecutive beta-strands, the central one containing the RNP1 motif, create a surface rich in aromatic and basic residues that are presumably involved in nucleic acid binding. Preferential binding of CspB to single-stranded DNA is observed in gel retardation experiments.

The DNA double helix is not a regular, featureless barberpole molecule. Different base sequences have their own special signature, in the way that they influence groove width, helical twist, bending, and mechanical rigidity or resistance to bending. These special features probably help other molecules such as repressors to read and recognize one base sequence in preference to another. Single crystal x-ray structure analysis is beginning to show us the various structures possible in the B-DNA family. The DNA decamer C-C-A-A-G-A-T-T-G-G appears to be a better model for mixed-sequence B-DNA than was the earlier C-G-C-G-A-A-T-T-C-G-C-G, which is more akin to regions of poly(dA).poly(dT). The G.A mismatch base pairs at the center of the decamer are in the anti-anti conformation about their bonds from base to sugar, in agreement with nuclear magnetic resonance evidence on this and other sequences, and in contrast to the anti-syn geometry reported for G.A pairs in C-G-C-G-A-A-T-T-A-G-C-G. The ordered spine of hydration seen earlier in the narrow-grooved dodecamer has its counterpart, in this wide-grooved decamer, in two strings of water molecules lining the walls of the minor groove, bridging from purine N3 or pyrimidine O2, to the following sugar O4'. The same strings of hydration are present in the phosphorothioate analog of G-C-G-C-G-C. Unlike the spine, which is broken up by the intrusion of amine groups at guanines, these water strings are found in general, mixed-sequence DNA because they can pass by unimpeded to either side of a guanine N2 amine. The spine and strings are perceived as two extremes of a general pattern of hydration of the minor groove, which probably is the dominant factor in making B-DNA the preferred form at high hydration.

XDSAPP is a Tcl/Tk‐based graphical user interface for the easy and convenient processing of diffraction data sets using XDS. It provides easy access to all XDS functionalities, automates the data processing and generates graphical plots of various data set statistics provided by XDS. By incorporating additional software, further information on certain features of the data set, such as radiation decay during data collection or the presence of pseudo‐translational symmetry and/or twinning, can be obtained. Intensity files suitable for CCP4, CNS and SHELX are generated.

The three-dimensional structure of the hy-

XDSAPPis an expert system and graphical user interface (GUI) for the automated processing of diffraction images using theXDSprogram suite and other programs. The latest major update and the extension of the program are presented here. The update includes new features, as well as improvements in the GUI and the underlying decision-making system.XDSAPPis freely available for academic users.