Guide to Protein Purification

861 Pages·1990·14.139 MB·English

by  John N. Abelson

Preface
The explosion of work and interest in molecular biology in recent
years has made protein purification something of a lost art, especially
among younger biochemists and molecular biologists. At the same time,
many of the more interesting biological problems have now reached a
stage that requires work with purified proteins and enzymes. This has led
to a situation in which many important studies stop at the demonstration
of a physiological effect, and are not carried through to an understanding
of the proteins responsible for the phenomenon. For these reasons a
methods manual dealing with all aspects of protein purification should be
a valuable addition to the Methods in Enzymology series and should be
extremely useful to the scientific community.
Although techniques for protein purification have been included in a
few volumes in this series in the past, this Guide brings together in one
source up-to-date procedures for purifying, characterizing, and working
with proteins and enzymes. The volume begins with introductory chap-
ters describing the rationale for studying proteins and enzymes with strat-
egies for their purification, is followed by contributions that familiarize
the reader with procedures for working with proteins and enzymes, and
proceeds to describe in detail methods for their purification and character-
ization. Useful immunological procedures and other techniques that aid in
the study of proteins are also included.
In addition to the methods articles that make up the bulk of the Guide,
a few retrospective chapters by eminent biochemists, which describe one
of their famous studies in order to give a feeling for the "art" of enzyme
purification that goes beyond techniques and mechanical procedures,
have been included.
The Guide is a self-contained volume covering all the important proce-
dures for purifying proteins, as well as other more specialized techniques.
However, to stay within the confines of a single volume, some details are
dealt with by reference to other works, but these have been kept to a
minimum. It is hoped that this volume will satisfy the needs of both the
novice in protein purification and the more experienced researcher.
YARRUM P. REHCSTUED
iiix
Contributors to Volume 182
are in numbers Article parentheses gniwollof names the of .srotubirtnoc
snoitailiffA are current. listed
PATRICK ARGOS (56), European Molecular Biochemistry, Albert Einstein College of
Biology Laboratory, 6900 Heidelberg, Medicine, Bronx, New York 16401
Federal Republic of Germany GARY L. FIRESTONE (52), Department of
JOHN S. BLANCHARD (4), Department of Molecular and Cell Biology and Cancer
Biochemistry, Albert Einstein College of Research Laboratory, University of Cali-
Medicine, Bronx, New York 16401 fornia at Berkeley, Berkeley, California
MARGARET K. BRADLEY (10), Department 02749
of Pathology, Dana-Farber Cancer Insti- STEPHEN C. FRANCESCONI (39), Depart-
tute and the Harvard Medical School, ment of Microbiology, University of Con-
Boston, Massachusetts 51120 necticut Health Center, Farmington,
ROMAN M. CHICZ (32), Department of Bio- Connecticut 23060
chemistry and Molecular Biology, Har- DAVID E. GARFIN (33, 35), Chemical Divi-
vard University, Cambridge, Massachu- sion, Research Products Group, Bio-Rad
setts 02138 Laboratories, Incorporated, Richmond,
CHRIS CIVALIER (39), Department of Micro- California 40849
biology, University of Connecticut Health PETER GEGENHEIMER (14), Departments
Center, Farmington, Connecticut 23060 of Botany and Biochemistry, University of
MILLARD CULL (12), Department of Bio- Kansas, Lawrence, Kansas 54066
chemistry, Biophysics and Genetics, Uni- CRAIG GERARD (40), Department of Pediat-
versity of Colorado Health Sciences Cen- rics, Harvard Medical School, Children's
ter, Denver, Colorado 80262 Hospital Medical Center, Boston, Massa-
AsIs DAS (9), Department of Microbiology, chusetts 51120
University of Connecticut Health Center, LALLAN GIRl (31), Quality Control Depart-
Farmington, Connecticut 23060 ment, Connaught Laboratories, Inc.,
MURRAY P. DEUTSCHER (3, 8, 57), Depart- Swiftwater, PA 07381
ment of Biochemistry, University of Con- MARINA J. GORBUNOFF (26), Graduate De-
necticut Health Center, Farmington, partment of Biochemistry, Brandeis Uni-
Connecticut 23060 versity, Waltham, Massachusetts 45220
JOHN DAVID DIGNAM (15), Department of MICHAEL G. HARRINGTON (37), Biology De-
Biochemistry, Medical College of Ohio, partment, California Institute of Technol-
Toledo, Ohio 43699 ogy, Pasadena, California 52119
BONNIE S. DUNBAR (34, 49--51), Depart- DONNA L. HARTLEY (20), Centre Interna-
ment of Cell Biology, Baylor College of tional de Recherche Daniel Carasso,
Medicine, Houston, Texas 03077 05329 Le Plessis-Robinson, Paris, France
SHLOMO EISENBERG (39), Department of LEONARD M. HJELMELAND (19, 21), De-
Microbiology, University of Connecticut partments of Ophthalmology and Biologi-
Health Center, Farmington, Connecticut cal Chemistry, School of Medicine, Uni-
23060 versity of California, Davis, Davis,
SASHA ENGLARD (22, 47), Department of California 61659
ix
X CONTRIBUTORS TO VOLUME 182
I. YRRAB (11), HOLLAND Department of Ge- tute, Massachusetts Institute of Technol-
netics, University of Leicester, Leicester ogy, Cambridge, Massachusetts 24120
LEI 7RH, England SELRAHC S. (12), MCHENRY Department of
B. L. REKCEROH (59), Department of Bio- Biochemistry, Biophysics and Genetics,
chemistry, Cornell University Medical University of Colorado Health Sciences
College, New York, New York 12001 Center, Denver, Colorado 80262
KENNETH C. INGHAM (23), Biochemistry MARK G. MCNAMEE (38), Department of
Laboratory, American Red Cross Hol- Biochemistry and Biophysics, University
land Laboratories, Rockville, Maryland of California, Davis, California 61659
55802 CARL R. MERRIL (36), Laboratory of Bio-
S. (13), JAZWlNSKI MICHAL Department of chemical Genetics, National Institute of
Biochemistry and Molecular Biology, Mental Health, Bethesda, Maryland
Louisiana State University Medical Cen- 29802
ter, New Orleans, Louisiana 21107
KIVIE EVADLOM (61), Department of Biol-
HPLAR C. DDUJ (46), Division of Biological ogy, University of California, Santa Cruz,
Sciences, University of Montana, Mis- Santa Cruz, California 46059
soula, Montana 21895
JUDITH M. (18), NEUGEBAUER Department
TREBOR M. (27), KENNEDY Membrex Incor- of Chemistry and Institute of Colloid and
porated, Garfield, New Jersey 62070 Surface Science, Clarkson University,
NADNERB KENNY 1( l), Department of Ge- Potsdam, New York 67631
netics, University of Leicester, Leicester
(48), OLLIS DAVID Department of Biochem-
LEI 7RH, England
istry, Molecular Biology and Cell Biology,
HITOMI KIMURA (34), Department of Bio- Northwestern University, Evanston, Illi-
chemistry, State University of New York nois 60208
at Stony Brook, Stony Brook, New York
STEVEN (29, OSTROVE 30), Davy McKee
49711
Corporation, Berkeley Heights, New Jer-
ARTHUR GREBNROK (1, 58), Department of sey 22970
Biochemistry, Stanford University, Stan-
JuRlS OZOLS (17, 44), Department of Bio-
ford, California 50349
chemistry, University of Connecticut
SAMOHT M. LAUE (42, 43), Department of
Health Center, Farmington, Connecticut
Biochemistry, University of New Hamp-
23060
shire, Durham, New Hampshire 03824
STUART LINN (2), Division of Biochemistry SELRAHC W. (53, PARKER 54), Department
of Medicine and Microbiology, Washing-
and Molecalar Biology, University of Cal-
ton University School of Medicine, St.
ifornia, Berkeley, Berkeley, California
Louis, Missouri 01136
02749
EDWARD A. MADDEN (16), Department of SAERDNA (11), PLOCKTHUN Gen-Zentrum
Biology, University of Indianapolis, Indi- derU niversitat Miinchen, Max-Planck-In-
anapolis, Indiana 46227 stitut flir Biochemie, D-8033 Martinsried,
FIONA A. O. NOTSRAM (20), Celltech Lim- Munich, Federal Republic of Germany
ited, Slough, Berkshire SL1 4EN, En- SAMOHT (7), POHL Abteilungfiir Molekulare
gland Neuroendokrinologie, Max-Planck-lnsti-
REHPOTSIRHC K. MATHEWS (41), Depart- tut fiir Experimentelle Medizin, 3400 G6t-
ment of Biochemistry and Biophysics, Or- tingen, Federal Republic of Germany
egon State University, Corvallis, Oregon FRED E. (32), REGNIER Department of Bio-
13379 chemistry, Purdue University, West La-
(45), PAUL MATSUDAIRA Whitehead lnsti- fayette, Indiana 47907
CONTRIBUTORS TO VOLUME 182 xi
DAVID G. RHODES (42, 43), Biomolecular Medicine, Bronx, New York 16401
Structure Analysis Center, Department of (16), BRIAN STORRIE Biochemistry Depart-
Radiology, University of Connecticut ment, Virginia Polytechnic Institute and
Health Center, Farmington, Connecticut State University, Blacksburg, Virginia
23O6O 06042
EDWARD F. ODNAMOSSOR (5, 24), Depart- ATSIRHC M. KCEHCSOTS (6), Department of
ment of BioStructure and Function, Uni- Medicine, Division of Dermatology, Vet-
versity of Connecticut Health Center, erans Administration, Nashville, Tennes-
Farmington, Connecticut 23060 see 37212
ERIc D. LEBEOWHCS (49), Department of SAMOHT C. (38), THOMAS Department of
Cell Biology, Baylor College of Medicine, Biochemistry and Biophysics, University
Houston, Texas 03077 of California, Davis, Davis, California
MAS SEIFTER (22, 47), Department of Bio- 61659
chemistry, Albert Einstein College of ESEREHT M. SNOMMIT (34, 51), Department
Medicine, Bronx, New York 16401 of Cell Biology, Baylor College of Medi-
SHERI M. (50), SKINNER Department of Cell cine, Houston, Texas 03077
Biology, Baylor College of Medicine, O. (59), TSOEAS Laboratory of Biological
Houston, Texas 03077 Chemistry, University of loannina Medi-
PAUL A. (41), SRERE Research Service, De- cal School, Ioannina, RaC 354 23 Greece
partment of Veteran Affairs, University of TTOCS S. (39), WALKER Department of Mi-
Texas Southwestern Medical Center, Dal- crobiology, University of Connecticut
las, Texas 61257 Health Center, Farmington, Connecticut
EARL R. STADTMAN (60), National Heart, 23060
Lung and Blood Institute, National Insti- YLLEHS (30), WEISS New Brunswick Scien-
tutes of Health, Bethesda, Maryland tific, Edison, New Jersey 81880
29802
NEHPETS WHITE (48), Department of Biol-
BORIS STEIPE (l ,)1 Gen-Zentrum der Un- ogy, Brookhaven National Laboratory,
iversitat Miinchen, Max-Planck-Institut Upton, New York 37911
fiir Biochemie, D-8033 Martinsried, Mu- ARDNAS D. (52), WINGUTH Department of
nich, Federal Republic of Germany Ophthalmology, Ocular Oncology Unit,
EARLE NEGAWLLETS (25, 28), Department University of California at San Francisco,
of Biochemistry, University of Iowa, Iowa San Francisco, California 34149
City, Iowa 52242
JOHN M. (55), WOZNEY Genetics Institute,
VINCENT S. (4), STOLE Department of Bio- Incorporated, Cambridge, Massachusetts
chemistry, Albert Einstein College of 04120
[1]
WHY PURIFY ENZYMES? 1
[1] Why Purify Enzymes?
By ARTHUR GREBNROK
"Don't waste clean thinking on dirty enzymes" is an admonition of
Efraim Racket's which is at the core of enzymology and good chemical
practice. It says simply that detailed studies of how an enzyme catalyzes
the conversion of one substance to another is generally a waste of time
until the enzyme has been purified away from the other enzymes and
substances that make up a crude cell extract. The mixture of thousands of
different enzymes released from a disrupted liver, yeast, or bacterial cell
likely contains several that direct other rearrangements of the starting
material and the product of the particular enzyme's action. Only when we
have purified the enzyme to the point that no other enzymes can be
detected can we feel assured that a single type of enzyme molecule directs
the conversion of substance A to substance B, and does nothing more.
Only then can we learn how the enzyme does its work.
The rewards for the labor of purifying an enzyme were laid out in a
series of inspirational papers by Otto Warburg in the 1930s. From his
laboratory in Bedin-Dahlem came the discipline and many of the methods
of purifying enzymes and with those the clarification of key reactions and
vitamin functions in respiration and the fermentation of glucose. War-
burg's contributions strengthened the classic approach to enzymology
inaugurated with Eduard Btichner's accidental discovery, at the turn of
this century, of cell-free conversion of sucrose to ethanol. One tracks the
molecular basis of cellular function--alcoholic fermentation in yeast, gly-
colysis in muscle, luminescence in a fly, or the replication of DNA--by
first observing the phenomenon in a cell-free system. Then one isolates
the responsible enzyme (or enzymes) by fractionation of the cell extract
and purifies it to homogeneity. Then one hopes to learn enough about the
structure of the enzyme to explain how it performs its catalytic functions,
responds to regulatory signals, and is associated with other enzymes and
structures in the cell.
By a reverse approach, call it ,lacissalcoen especially popular in re-
cent decades, one first obtains a structure and then looks for its function.
The protein is preferably small and stable, and has been amplified by
cloning or is commercially available. By intensive study of the protein and
homologous proteins, one hopes to get some clues to how it functions. As
the popularity of the neoclassical approach has increased, so has there
Copyright © 1990 by Academic Press, Inc.
METHODS IN ENZYMOLOGY, VOL. 281 All rights of reproduction in any form reserved.
2 METHODS IN ENZYMOLOGY [1]
been a corresponding decrease in interest in the classical route: pursuit of
a function to isolate the responsible structure.
Implicit in the devotion to purifying enzymes is thef aith of a dedicated
biochemist of being able to reconstitute in a test tube anything a cell
can do. In fact, the biochemist with the advantage of manipulating the
medium: pH, ionic strength, etc., by creating high concentrations of
reactants, by trapping products and so on, should have an easier time
of it. Another article of faith is that everything that goes on in a cell is
catalyzed by an enzyme. Chemists sometimes find this conviction difficult
to swallow.
On a recent occasion I was told by a mature and well-known physical
chemist that what fascinated him most in my work was that DNA replica-
tion was catalyzed by enzymes ! This reminded me of a seminar I gave to
the Washington University chemistry department when I arrived in St.
Louis in 1953. I was describing the enzymes that make and degrade orotic
acid, and began to realize that my audience was rapidly slipping away.
Perhaps they had been expecting to hear about an organic synthesis of
erotic acid. In a last-ditch attempt to retrieve their attention, I said loudly
that every chemical event in the cell depends on the action of an enzyme.
At that point, the late Joseph Kennedy, the brilliant young chairman,
awoke: "Do you mean to tell us that something as simple as the hydration
of carbon dioxide (to form bicarbonate) needs an enzyme?" The Lord had
delivered him into my hands. "Yes, Joe, cells have an enzyme, called
carbonic anhydrase. It enhances the rate of that reaction more than a
million fold."
Enzymes are awesome machines with a suitable level of complexity.
One may feel ill at ease grappling with the operations of a cell, let alone
those of a multiceUular creature, or feel inadequate in probing the fine
chemistry of small molecules. Becoming familiar with the personality of
an enzyme performing in a major synthetic pathway can be just right. To
gaint his intimacy, the enzyme must first be purified to near homogeneity.
For the separation of a protein species present as one-tenth or one-hun-
dredth of %1 of the many thousands of other kinds in the cellular commu-
nity, we need to devise and be guided by a quick and reliable assay of its
catalytic activity.
No enzyme is purified to the point of absolute homogeneity. Even
when other proteins constitute less than %1 of the purified protein and
escape detection by our best methods, there are likely to be many millions
of foreign molecules in a reaction mixture. Generally, such contaminants
do not matter unless they are preponderantly of one kind and are highly
active on one of the components being studied.
[1] YHW PURIFY ?SEMYZNE 3
Only after the properties of the pure enzyme are known is it profitable
to examine its behavior in a crude state. "Don't waste clean thinking on
dirty enzymes" is sound dogma. I cannot recall a single instance in which
I begrudged the time spent on the purification of an enzyme, whether it
led to the clarification of a reaction pathway, to discovering new en-
zymes, to acquiring a unique analytical reagent, or led merely to greater
expertise with purification procedures. So, purify, purify, purify.
Purifying an enzyme is rewarding all the way, from first starting to free
it from the mob of proteins in a broken cell to having it finally in splendid
isolation. It matters that, upon removing the enzyme from its snug cellular
niche, one cares about many inclemencies: high dilution in unfriendly
solvents, contact with glass surfaces and harsh temperatures, and expo-
sure to metals, oxygen, and untold other perils. Failures are often attrib-
uted to the fragility of the enzyme and its ready denaturability, whereas
the blame should rest on the scientist for being more easily denatured.
Like a parent concerned for a child's whereabouts and safety, one cannot
leave the laboratory at night without knowing how much of the enzyme
has been recovered in that day's procedure and how much of the contami-
nating proteins still remain.
To attain the goal of a pure protein, the cardinal rule is that the ratio of
enzyme activity to the total protein is increased to the limit. Units of
activity and amounts of protein must be strictly accounted for in each
manipulation and at every stage. In this vein, the notebook record of an
enzyme purification should withstand the scrutiny of an auditor or bank
examiner. Not that one should ever regard the enterprise as a business or
banking operation. Rather, it often may seem like the ascent of an un-
charted mountain: the logistics like those of supplying successively higher
base camps. Protein fatalities and confusing contaminants may resemble
the adventure of unexpected storms and hardships. Gratifying views
along the way feed the anticipation of what will be seen from the top. The
ultimate reward of a pure enzyme is tantamount to the unobstructed and
commanding view from the summit. Beyond the grand vista and thrill of
being there first, there is no need for descent, but rather the prospect of
even more inviting mountains, each with the promise of even grander
views.
With the purified enzyme, we learn about its catalytic activities and its
responsiveness to regulatory molecules that raise or lower activity. Be-
yond the catalytic and regulatory aspects, enzymes have a social face that
dictates crucial interactions with other enzymes, nucleic acids, and mem-
brane surfaces. To gain a perspective on the enzyme's contributions to
the cellular economy, we must also identify the factors that induce or
4 METHODS IN ENZYMOLOGY [1]
repress the genes responsible for producing the enzyme. Tracking a meta-
bolic or biosynthetic enzyme uncovers marvelous intricacies by which a
bacterial cell gears enzyme production precisely to its fluctuating needs.
Popular interest now centers on understanding the growth and devel-
opment of flies and worms, their cells and tissues. Many laboratories
focus on the aberrations of cancer and hope that their studies will furnish
insights into the normal patterns. Enormous efforts are also devoted to
AIDS, both to the virus and its destructive action on the immune system.
In these various studies, the effects of manipulating the cell's genome and
the actions of viruses and agents are almost always monitored with intact
cells and organisms. Rarely are attempts made to examine a stage in an
overall process in a cell-free system. This reliance in current biological
research on intact cells and organisms to fathom their chemistry is a
modern version of the vitalism that befell Pasteur and that has permeated
the attitudes of generations of biologists before and since.
It baffles me that the utterly simple and proven enzymologic approach
to solving basic problems in metabolism is so commonly ignored. The
precept that discrete substances and their interactions must be under-
stood before more complex phenomena can be explained is rooted in the
history of biochemistry and should by now be utterly commensensical.
Robert Koch, in identifying the causative agent of an infectious disease,
taught us a century ago that we must first isolate the responsible microbe
from all others. Organic chemists have known even longer that we must
purify and crystallize a substance to prove its identity. More recently in
history, the vitamin hunters found it futile to try to discover the metabolic
and nutritional roles of vitamins without having isolated each in pure
form. And so with enzymes it is only by purifying enzymes that we can
clearly identify each of the molecular machines responsible for a discrete
FIG. .1 Personalized license plate expressing a commitment to enzymology.
[1] YHW YFIRUP .9SEMYZNE 5
metabolic operation. Convinced of this, one of my graduate students
expressed it in a personalized license plate (Fig. 1).
Acknowledgment
This article borrows extensively from "For the Love of Enzymes: The Odyssey of a
Biochemist," Harvard University Press, .9891
[2] GENERAL STRATEGIES AND CONSIDERATIONS 9
[2] Strategies and Considerations for Protein Purifications
By LINN STUART
The budding enzymologist is generally surprised by the time necessary
to develop a protein purification procedure relative to the time required to
accumulate information once the purified protein is available. While there
is no magic formula for designing a protein purification, some forethought
can help to expedite the tedious job of developing the purification scheme.
This chapter is designed to point out some considerations to be under-
taken prior to stepping up to the bench. Once at the bench, the subsequent
chapters of this book as well as two other recent publications concerning
enzyme purification 2,1 should serve as a guide.
Preliminary Considerations
What Is the Protein To Be Used For
In these days of the biotechnology revolution, the required amount of
purified protein may vary from a few micrograms needed for a cloning
endeavor to several kilograms required for an industrial or pharmaceuti-
cal application. Therefore, a very major consideration is the amount of
material required. One should be aware of the scale-up ultimately ex-
pected, and the final scheme should be appropriate for expansion to those
levels. There are very real limitations to how far a procedure can be
scaled up. These limitations are brought about not only by considerations
of cost and availability of facilities, but also by physical constraints of
such factors as chromatographic resin support capabilities and electro-
phoresis heating factors. As outlined below, individual steps of the proce-
dure should flow from high-capacity/low-cost techniques toward low-
capacity/high-cost ones. Nonetheless, in some cases two procedures may
be required: for example, one to obtain microgram quantities for cloning
and a second to produce kilogram amounts of the cloned material. The
protein chemist should remain flexible for adopting new procedures when
such changes are warranted.
Another consideration is whether the protein must be active (an en-
zyme, a regulatory protein, or an antibody, for example), whether it must
R. K. Scopes, "Protein Purification, Principles and Practice," 2nd Ed. Springer-Verlag,
New York, 1987.
2 R. Burges, ed., "Protein Purification, Micro to Macro." Alan R. Liss, New York, 1987.
thgirypoC © 0991 yb cimedacA Press, .cnI
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