Protein purification methods: A practical approach

~]OOK

Prediction of Protein Structure and the Principles of Protein Conformation edited by GeraldD. Fasman,PlenumPress, 1989. S95.00(xiii + 798 pages) ISBN0 306 43131 9 It is expected (or at least hoped) that within the next five to ten years we shall know the DNA sequences of the majority of genes from a variety of organisms, including Escherichia, Saccharomyces, Arabidopsis, Caenorhabditis, Drosophila, Mus and Homo. One of the prime reasons for attempting large-scale physical mapping and sequencing projects must be to infer the sequences of the encoded proteins. The exciting part of these genome projects is therefore going to be the challenge presented to workers on protein structure and function. Given several hundred thousand new putative protein sequences, can anything useful lye said on the basis of existing knowledge or will a decade of sequencing be followed by centuries of protein structure determination? There is some feeling of optimism that the task will not be too daunting because similar themes will recur (Doolittle). However, there seems to be no current support for the view that a universal protein folding law will be discovered to enable structure to be derived from sequence. A problem with the field of protein structure prediction, as with RNA folding, is that one can make intellectually and aesthetically pleasing predictions in the total absence of testability, The long sought after protein folding rules can only deal with particular situations and not with generalities, In his introduction, the editor of this book wisely restricts himself to a narrower goal: the possibility' of designing new proteins by small modifications to those whose structure and function is known. Sitedirected mutagenesis and expression systems make this feasible. The changes in properties can be

[~EVIEWS

predicted in advance and the success of these predictions can be evaluated by experimental determination of the properties of the protein. This is a monumental work of 798 pages by 36 authors, arranged in 20 chapters that range from the particular (the photochemical reaction center of Rbodopseudomonas viridis; Deisenhofer el al.) through wider subjects (virus structure; Stubbs) to the very general (e.g. stabilization energies of protein conformation; Albers), The treatmerit is extremely comprehensive and is in a review style rather than being introductory or an overview. In places this is carried too far and the prose degenerates into a mechanical outpouring of references. As an outsider to the field, I particularly enjoyed the chapter by Jane and David Richardson on 'Principles and patterns of protein conformation', which gives a good feeling for the principles that enable one to appreciate the essence of protein structures. There is an exhaustive history of protein

structure prediction by Fasman, with sources of programs and data banks, The whole range of secondary structure prediction methods is dealt with, as are hydrophobicity and tertiary structure prediction. The treatment is not confined to globular proteins. and there are chapters on membrane proteins. Having waded through the book I was left with a slight feeling of disappointment that the promise of the editor's preface about the ability to design proteins at will appears not to have been fulfilled. There is an awful lot about prediction and not much about results of experiments to modify proteins and determine the changes to their structures by crystallography or nuclear magnetic resonance, measure chariged physical properties and measure biological activities in such engineered proteins.

M.J.BISHOP MRCMolecularGeneticsUnit,HillsRoad, CambridgeCB22QH, UK.

enlisted - and the book is therefore scattered with many wise remarks and much good advice, especially on the types of apparatus available the price paid is the absence of an overview. Nowhere does the book clearly state the principles of separations. As a result, readers are given recipes rather than taught to think for themselves. So they do not learn why electrophoresis is usually better analytically than preparatively, why the ionic concentration of a buffer used in ion exchange should be sufficiently high that it is not easily changed by the sample, or why gradients rather than isocratic chromatography are usually necessary for proteins. Ammonium sulphate precipitation is rightly suggested for an early step in a purification, but it is not explained that high salt concentration prevents (by ion exchange) a precipitate from adsorbing a protein from solution, as happens easily in other precipitations. There is no mention of the need to control the concentration of the protein being purified if it is to be precipitated at reproducible -

Protein Purification Methods: A Practical Approach edited by E.L.V.Harrisand S. kngal, IRLPress at OxfordUniversityPress, 1989.£30.00 hbk, £20.00 pbk (x~'i+ 317 pages)ISBN0 19 %3OO38

Protein Pur{fication Methods is divided into two sets of three chapters: the first three are on initial planning of a purification, on extracting a protein and clarifying the extract, and on concentrating the extract; three more follow on the main methods of purification, those based on structure, on specific binding properties of the protein, and on molecular size, Individual chapters - and even small sections within them - have different authors. While this structure has tile advantage that authors with wide varieties of expertise can be rt(; MARCH1991 VOL. 7 NO. 3 I()(

WOOK salt concentrations (M. Dixon [no relation!l and E.C. Webb Adv. Protein Chem. 16, 197-219, 1961). The chapter on using the specific binding properties of the protein deals only with affinity chromatography, that is, the use of a specifically made adsorbent and non-

Gene Probes (Methods in Neuroscience, Vol. 1) edited by PM. Conn, AcademicPress, 1989. 561.00 (xiii + ,~64pages) ISBN0 12 185251 2 The application of molecular biology techniques to the nervous system has provided substantial advances in our understanding of the molecular basis of neural developmerit and function. A volume dealing with the practical aspects of the main molecular techniques, with examples of their use in the nervous system, is therefore timely and potentially very useful for neuroscientists wishing to exploit these powerful techniques. Unfortunately, Gene Probes" is

[]~EVIEWS

specific elution. The cost and time savings of the reverse procedure binding to a cheap ion exchanger, and eluting with a specifically bound ligand (see, for example, R.K. Scopes, Biochem.J. 161, 253-263 and 265-277, 1977) - are not mentioned. A minor, but irritating, fault is the failure to follow internationally recognized standards for symbols and terms, so that absorbance is called OD or extinction, symbols for physical quantities are often not italicized, daltons are symbolized 'd' not 'Da' (symbols for units have capital letters if based on proper names) and even the grammar and spelling are poor in places. The Et3N+ group is called triethylamino (the amino group H2N can only have two substituents) rather than triethylammonio. Errors spoil some formulae (an error in the name of one of the constituent sugars of agarose means that the reader cannot check what the structure should be). Unjustified assertions are made, for example that cacodylate is toxic (it may be a poor idea to use it as a buffer because of its reduction by thiols and its expense), that

cyanogen bromide can release HCN (rather than cyanate), that an oxidizing environment can cause denaturation (denaturation is a noncovalent change, which may well lead to oxidation by unmasking interior groups). We read that phos phate buffers should be avoided in freeze-drying, because their pH drops on cooling (phosphate, like any other nonvolatile buffer, will cause partial thawing). Similarly it is stated that dithiothreitol 'lasts longer' than mercaptoethanol in 1)uffcrs, without an explanation of why it is a more powerful reducing agent, and that no great excess is needed. Despite the above list (which could be extended) of failures to explain principles, the authors have given a good survey of the best methods of purification and of the types of apparatus available, and they have made many wise remarks on the use of the methods and design of purifications. The book may therefore be of considerable use.

rather disappointing, not because of any deficiencies in the individual chapters - most of which are excellent - but because of excessive repetition and the omission of several major techniques. For example, four chapters address the use of the X e n o p u s oocyte for the expression and cloning of neuro receptor and ion channel genes. The authors of each chapter inde pendently provide an introduction to this system, describing its advantages, and give comprehensive details of experimental procedures. In addition to the repetition this entails, the protocols provided in each chapter are different. Since there is no overall discussion of these techniques, the reader is left a little perplexed. More logically, this section would have contained a single introduction and review of the subject followed by a comparative description and discusskm of the methods used for mRNA prep aration, oocyte removal, micro-

injection and assay procedures. Redundancy is particularly con spicuous in the 13 self-contained chapters on in situ hybridization. Although digoxigenin-labelled and radiolabelled probes (cRNA and oligonucleotide) are covered m different chapters, many of the chap lcrs are superfluous. Surprisingly, not all of the chapters placc sufficient emphasis on the importance of control experiments, such as northern blots (if possible), to verilk' the specificity of the probe before use in situ. A notable omisskm is any detailed discussion of northern blotting, an important and widclv used technique in studies of gene expression in the nervous system, Although the use of RNase protcc tion assays to detect mRNA precur sots in the nucleus is discussed, no mention is made of the use of reverse transcription follox~cd b\ amplifk'atkm of the first strand eDNA by the polymerasc chain

T1G MARCH1991 VOL.7 NO. 3

H.B.E DIXON Department qf Biocbernisto,. l'nh,ersi(r o/ Cambridge, Cambndge CB2 l~t{'i I I<.