Protein Expression A Practical Approach

by Higgins, S. J.; Hames, B. D.

• ISBN13: 9780199636235
• ISBN10: 0199636230
• Format: Paperback
• Copyright: 1999-07-29
• Publisher: Oxford University Press

Summary

Part of a mini-series on the synthesis and processing of proteins, this volume details the expression of cloned DNA and RNA templates in all of the major in vivo and in vitro systems. The in vivo systems covered are cultured mammalian cells, the yeasts Sacchoromyces cerevisiae and Pichia pastoris, baculovirus, Xenopus oocytes, and prokaryotic cells. It also covers cell-free systems of both eukaryotes and prokaryotes, including prokaryotic systems that offer coupled transcription-translation. The volume concludes with a chapter on monitoring protein expression.

Table of Contents

List of contributors	xv
Abbreviations	xvii
Protein expression in mammalian cells	1	(28)
Marlies Otter-Nilsson
Tommy Nilsson
Introduction	1	(1)
Viral and plasmid vectors	1	(9)
Semliki forest virus	1	(3)
Vaccinia virus	4	(3)
Retroviral vectors	7	(1)
Plasmid pCMUIV	8	(2)
Plasmid pSRα	10	(1)
Transient and stable transfection methods	10	(12)
Calcium phosphate	10	(3)
DEAE--dextran	13	(1)
Lipid-mediated transfection	14	(1)
Electroporation	15	(1)
Microinjection	16	(2)
Stable transfection and selection	18	(2)
Inducible protein expression in stable cell lines	20	(2)
Detection of expressed protein	22	(7)
GFP as a tool in protein expression	22	(1)
Epitope tags	23	(2)
References	25	(4)
Expression in Xenopus oocytes and cell-free extracts	29	(32)
Glenn M. Matthews
Introduction	29	(2)
Translation in oocytes	29	(1)
Xenopus egg extracts	30	(1)
Maintaining Xenopus laevis stocks	30	(1)
Xenopus oocyte microinjection	31	(15)
Equipment	31	(1)
Obtaining and culturing oocytes	32	(4)
mRNA	36	(2)
Use of the injector	38	(2)
Microinjection	40	(1)
Expression from microinjected DNA	41	(1)
Radioactive labelling	41	(2)
Analysis of radioactive translation products	43	(2)
Fractionation of oocytes	45	(1)
Preparation and use of Xenopus egg cell-free extracts	46	(15)
Equipment	46	(2)
Xenopus eggs	48	(1)
Preparation of extract	49	(4)
In vitro translation using Xenopus cell-free extracts	53	(2)
Analysis of translation products	55	(3)
References	58	(3)
Expressing cloned genes in the yeasts Saccharomyces cerevisiae and Pichia pastoris	61	(40)
Mick F. Tuite
Jeff J. Clare
Mike A. Romanos
Introduction	61	(2)
Saccharomyces cerevisiae expression systems	63	(20)
Plasmid-based vectors	63	(3)
Transformation of S. cerevisiae	66	(2)
Choice of strain of S. cerevisiae	68	(1)
Transcription and translation of heterologous genes and cDNAs	69	(3)
Directing the extracellular synthesis of heterologous proteins	72	(4)
Analysis of heterologous gene expression	76	(7)
Pichia pastoris expression systems	83	(18)
Introduction	83	(1)
Expression strategies	84	(3)
Host--vector systems and transformation methods for P. pastoris	87	(6)
Analysis of DNA from P. pastoris transformants	93	(2)
Induction of foreign protein expression in P. pastoris	95	(4)
References	99	(2)
Baculovirus expression systems	101	(28)
Claire L. Merrington
Linda A. King
Robert D. Possee
Introduction	101	(1)
Baculovirus life cycle	102	(1)
Insect cell culture	103	(6)
Baculovirus expression vectors	109	(4)
Manipulating the baculovirus genome	109	(1)
Baculovirus transfer vectors	109	(3)
Preparation of recombinant transfer vectors	112	(1)
Preparation of recombinant virus	113	(5)
Optimizing the selection of recombinant virus	113	(2)
Co-transfection of insect cells with linearized viral DNA and recombinant transfer vectors	115	(2)
Identification and purification of recombinant viruses	117	(1)
Characterization of recombinant virus DNA	118	(2)
Analysis of protein synthesis in virus-infected cells	120	(2)
Post-translational modification of proteins synthesized using the baculovirus expression system	122	(1)
Scaling up recombinant protein production	123	(1)
Alternative methods for producing recombinant baculoviruses	124	(1)
Baculovirus--yeast system	124	(1)
Bacmid system	125	(1)
Future developments of the baculovirus expression system	125	(4)
References	125	(4)
Protein synthesis in eukaryotic cell-free systems	129	(40)
Mike J. Clemens
Ger J. M. Pruijn
Introduction	129	(1)
Preparation of messenger RNAs	130	(6)
Precautions against RNase-mediated degradation	130	(1)
Preparation of intact RNA from ribosomal and polysomal fractions	131	(2)
Oligo(dT) affinity chromatography for isolation of poly(A)+ mRNA	133	(1)
Isolation of individual mRNA species	133	(1)
Transcription of mRNA in vitro	134	(2)
The reticulocyte lysate cell-free translation system	136	(10)
Preparation and storage of reticulocyte lysate	136	(4)
Assays of protein synthesis in reticulocyte lysates	140	(5)
Advantages and disadvantages of the reticulocyte lysate system	145	(1)
The wheat germ cell-free translation system	146	(4)
Sources of wheat germ	147	(1)
Preparation of wheat germ extracts	147	(1)
Assays of protein synthesis in wheat germ extracts	148	(1)
Advantages and disadvantages of the wheat germ system	149	(1)
Cell-free translation systems from other eukaryotic cell types	150	(1)
Methods for analysis of translation products	151	(5)
Radiosotopic methods	151	(1)
Chemiluminescence	152	(1)
Immunoprecipitation of translation products	153	(2)
Ligand binding assays	155	(1)
In vitro synthesis of membrane and secretory proteins	155	(1)
Specialized procedures	156	(13)
Synthesis of biotinylated proteins	156	(1)
Coupled in vitro transcription--translation systems	157	(1)
Cap-dependent versus internal initiation of translation	157	(1)
Assays for post-translational processing	158	(7)
The protein truncation test	165	(1)
Acknowledgements	165	(1)
References	165	(4)
Prokaryotic in vivo expression systems	169	(32)
Edward R. Appelbaum
Allan R. Shatzman
Introduction	169	(1)
General considerations in selecting an E. coli expression system	169	(6)
Choosing between E. coli and other expression systems	169	(2)
Improving the level of expression	171	(1)
Improving the solubility of a protein expressed in E. coli	172	(2)
Expression of heterologous proteins as fusion proteins or with protein tags	174	(1)
Nature of the N-terminus of the heterologous protein	175	(1)
Features of E. coli expression systems	175	(3)
Promoters and other transcription regulatory elements	175	(2)
Translation initiation and termination signals	177	(1)
Host strain	178	(1)
Protocols for expression: general comments	178	(2)
Expression, detection, and purification of a His6-tagged protein	180	(16)
Construction of the recombinant vector and transformation of host cells	180	(7)
Expression of the heterologous sequence	187	(2)
Analysis of expression of the heterologous protein	189	(4)
Purification of His6-tagged proteins	193	(3)
Expression of heterologous proteins in a secretion system	196	(3)
Sources of information on expression systems	199	(2)
Acknowledgements	199	(1)
References	199	(2)
Cell-free coupled transcription-translation systems from Escherichia coli	201	(24)
Gisela Kramer
Wieslaw Kudlicki
Boyd Hardesty
Background	201	(2)
Bacterial cell-free expression systems	201	(1)
Usefulness of cell-free coupled transcription--translation systems	202	(1)
Preparation of extracts and components for coupled transcription--translation systems	203	(8)
Preparation of the bacterial cell-free extract (S30)	203	(2)
Construction and preparation of plasmids	205	(3)
Preparation of RNA polymerases	208	(2)
Preparation of the low molecular weight mix (LM) for coupled transcription--translation	210	(1)
The coupled transcription--translation assay	211	(8)
The basic assay	211	(2)
Analysis of the product formed in the coupled transcription-translation assay	213	(6)
Modified coupled transcription-translation assays	219	(2)
Further developments	221	(4)
Acknowledgements	222	(1)
References	222	(3)
Monitoring protein expression	225	(42)
John Colyer
Introduction	225	(2)
General considerations	225	(1)
Basic strategies for monitoring protein expression	226	(1)
Immunodetection of protein expression	227	(30)
Considerations affecting the choice of antibody	227	(3)
Immunodot blots	230	(2)
Western blotting	232	(6)
Immunodetection of proteins on dot blots and Western blots	238	(9)
Pulse-chase labelling and immunoprecipitation of proteins	247	(7)
Examination of protein expression by immunomicroscopy	254	(3)
Monitoring of protein expression by epitope tagging	257	(2)
Surrogate reporter systems for monitoring protein expression	259	(8)
General principles	259	(1)
Quantification of protein X expression using the CAT reporter assay	260	(2)
Histological examination of protein expression using GUS reporter activity	262	(1)
Monitoring expression and cellular location using GFP	262	(2)
Acknowledgements	264	(1)
References	264	(3)
Appendix	267	(6)
Index	273