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Current Activities in Protein Engineering

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Current Activities in Protein Engineering
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Genetic engineering was a dual challenge for the eminent protein scientist Christian Anfinsen. He „was a thoughtful critic of the potential misuses of biotechnology and genetic engineering at a time when many of his colleagues were swept up by their promise“[1]. And he remained skeptical towards its ability to synthesize all kinds of proteins. He rather emphasized the prospects of classical protein chemistry. „At the same time while this DNA recombinant methodology has improved so enormously, there has been in a sort of quiet, secret way a great deal of improvement in the methodology of protein chemistry, particularly in the organic synthesis of peptides, even quite large ones,“ he remarks at the beginning of this lecture and briefly mentions the advances in protein purification technologies before discussing current protein engineering activities. „We have all been using the Merrifield solid phase method mostly“, says Anfinsen, paying his respects to Bruce Merrifield who had received the Nobel Prize in Chemistry just two years before in 1984 for his invention of solid phase peptide synthesis, which had culminated in the synthesis of the enzyme ribonuclease A with its 124 amino acids in 1969. Anfinsen also reminds his audience of „the fact that the purest polypeptide made synthetically was made entirely by hand that is to say not machines“. This is a tribute to Erich Wünsch who succeeded with his group in Munich in synthesizing the hormone glucagon with its 29 amino acids in 1967.In vitro peptide synthesis - as opposed to in vivo synthesis through genetic engineering - is difficult because each amino has two functional groups, which can enter into a peptide bond. To synthesize proteins in a controlled manner, i.e. in an intended sequence, functional groups that shall not be involved in the reaction must be shielded by protecting groups. In liquid-phase synthesis, the separation procedures, which are therefore necessary after each synthetic step, cause a considerable loss of product and diminish the peptide yield dramatically. Only small peptides with less than ten amino acids can be produced this way. The solid-phase procedure facilitates synthesis and increases its yield by attaching the first amino acid through its C-terminus to a solid polymer. This method is suitable for automation. The N-terminus of the growing peptide chain is protected by either t-boc (tert-butyloxycarbonyl) or Fmoc (9-fluorenylmethyloxycarbonyl). Anfinsen briefly discusses the characteristics of both groups and then suggests „another extension of protein synthesis“.He calls this method „stitching“ and means „sewing together large pieces“ of proteins to synthesize an even larger one. He reports how in his lab at the Johns Hopkins University the stitching technique had already been used „with considerable success“ when applied to pieces of staphylococci nuclease. He reports on similar experiments with thioredoxin. „If one could make large peptide fragments synthetically, it’s possible to stitch them together and regenerate the original protein“. Appropriate chemical site-directed mutagenesis could support this process. Anfinsen’s suggestion may have sounded anachronistic in the decade that witnessed the launch of the first genetically engineered insulin and other recombinant drugs. Yet even the genetic code has its limitations and not all proteins or their analogues can be engineered by harnessing the forces of bacteria, yeast and the like. Chemical synthesis is here to stay. Anfinsen’s suggestions were far-sighted, foreshadowing, for example, the introduction of native chemical ligation as a means of synthesizing large proteins in the mid-1990s. Joachim Pietzsch[1] The U.S. National Library of Medicine: Profiles in Science: The Christian B. Anfinsen Papers; http://profiles.nlm.nih.gov/ps/retrieve/Narrative/KK/p-nid/19
PressureNobeliumMeeting/Interview
Rekombinante DNSProteinWursthülleMeeting/Interview
Rekombinante DNSSecretionProteinMeeting/Interview
Lamb and muttonMagmaDNAProteinSynthetic oilWater purificationMeeting/Interview
ValineHigh-performance liquid chromatographyIonenaustauschAageBiotechnologyMeeting/Interview
Columbia RecordsMaterials scienceErdölraffinationWursthülleSynthetic oilMeeting/Interview
LigandSetzen <Verfahrenstechnik>WalkingMixtureMaterials scienceSynthetic oilErdölraffinationMeeting/Interview
Water purificationProteinPeptideMeeting/Interview
BiosynthesisAmino acidSurface finishingMeeting/Interview
HydrocarboxylierungOligomereAmineMethoxygruppeBiomolecular structureMeeting/Interview
TryptophanPeptide synthesisPeptideBiosynthesisOligomereMeeting/Interview
PeptideMeeting/Interview
MachinabilityBiosynthesisOrganische ChemieProteinMeeting/Interview
Separation processProteinSystemic therapyMeeting/Interview
ProteinNucleaseMeeting/Interview
ThiolgruppeLysineDisulfideTryptophanSetzen <Verfahrenstechnik>Meeting/Interview
ErdrutschNucleaseMoleculeProteinfaltungMeeting/Interview
Chemical structureChain (unit)Meeting/Interview
Helicität <Chemie>ErdrutschC-terminusBeta sheetMeeting/Interview
TrypsinMoleculeCalciumMeeting/Interview
Conformational isomerismTrypsinLysineThymidinIonenbindungMeeting/Interview
HydroglimmerLysineElectronic cigaretteChemical structureMeeting/Interview
IonenbindungProteinChemical structurePeptideMeeting/Interview
WaterGlycerinTrypsinCalciumChemical reactionThiamindiphosphatBiosynthesisMeeting/Interview
Systemic therapyIonenbindungMoleculeActivity (UML)Meeting/Interview
Synthetic oilErdrutschDerivative (chemistry)ChemistryTrypsinPosttranslational modificationMeeting/Interview
Table wineHydroxybuttersäure <gamma->Activity (UML)Residue (chemistry)IonenbindungAmino acidCalciumSubstitutionsreaktionAtomMeeting/Interview
CarboxylierungCalciumCarboxylateActivity (UML)Glutamic acidProteinAspartic acidMeeting/Interview
ArginineResidue (chemistry)Multiprotein complexActivity (UML)PhosphateMoleculeChain (unit)Meeting/Interview
DNAErdrutschChemistryMutageneseMeeting/Interview
GenotypeNucleaseActivity (UML)Amino acidMutageneseMoleculeMeeting/Interview
ProteinAnomalie <Medizin>MoleculeMeeting/Interview
Data conversionSetzen <Verfahrenstechnik>Functional groupDisulfidbrückeWine tasting descriptorsMeeting/Interview
Chemical structureProteinArginineResidue (chemistry)Amino acidMeeting/Interview
Spawn (biology)Radioactive decayMan pageFluorescenceMeeting/Interview
MoleculeBoyle-Mariotte-GesetzAmino acidTryptophanMeeting/Interview
ProteinChemistryMeeting/Interview
TryptophanBreed standardConformational isomerismPhenylalanineMeeting/Interview
ArginineResidue (chemistry)PeptideChain (unit)CitronensäureLysineMeeting/Interview
MoleculeActivity (UML)TrypsinMeeting/Interview
PressureAmino acidElektronentransferTryptophanFluorescenceC-terminusMeeting/Interview
PeptideMeeting/Interview
PeptideDNAProteinMeeting/Interview
BiosynthesisPeptideMeeting/Interview
TrypsinCaffeineCarboxypeptidase BHigh-performance liquid chromatographyMeeting/Interview
C-terminusFunctional groupAreaEnzymeMeeting/Interview
Ene reactionSynthetic oilImpfung <Chemie>ProteinChemical structureMeeting/Interview
Helicität <Chemie>NucleolusMoleculeAreaProteinBeta sheetAlpha particleMeeting/Interview
BromcyanSolutionChemical structureSpaltflächeResidue (chemistry)Meeting/Interview
Antibodies (film)MoleculeMeeting/Interview
PressureNucleaseChemical structureNucleolusThermoformingMeeting/Interview
Injection (medicine)AntigenAntibodies (film)MixtureNucleaseMeeting/Interview
Columbia RecordsSphäroproteineAntibodies (film)Surface scienceNucleaseSystemic therapyActive siteMeeting/Interview
Antibodies (film)PeptideMeeting/Interview
PeptideMeeting/Interview
Binding energyAntibodyProteinAntibodies (film)NucleaseActivity (UML)Meeting/Interview
NucleaseProteinThermoformingActivity (UML)Chemical structureMeeting/Interview
Binding energyAntibodies (film)Impfung <Chemie>Synthetic oilMeeting/Interview
LysozymePeptide sequenceMixtureFreies ElektronImpfung <Chemie>Synthetic oilAntibodies (film)AageMeeting/Interview
ProteinOligomereImpfung <Chemie>Meeting/Interview
Impfung <Chemie>Neutralization (chemistry)Meeting/Interview
River mouthChemical structureAntibodies (film)Meeting/Interview
NobeliumStuffingImpfung <Chemie>PeptideLactitolMeeting/Interview
Transcript: English(auto-generated)
Thank you, parents, for your kind introduction.
I thought in this lecture that I would take the opportunity to show you all that, in spite of people winning Nobel Prizes and so on, that they can still dress in a more or less civilized way, so I've worn a tie and jacket.
So, I should also mention that I've changed the title of my speech, and for the next half hour I intend to discuss matters of nuclear power, human rights, nuclear weapons, and the like.
I really think they'd rather have this, you know. Okay. What I will talk to you about is not so much a large amount of detailed information, although I will make illustrations, of course, from our own work and from other people's work,
but a feeling that I personally and many other people also have about the directions in which the preparation of proteins will go. The development of the DNA recombinant methodology, of course, has made it
possible in many cases to prepare large molecules, proteins, hormones, et cetera, and this is now a tremendous industry throughout the world and very successful. At the same time, while this DNA recombinant methodology has improved so enormously, there has been, in a sort of quiet, secret way,
a great deal of improvement in the methodology of protein chemistry, particularly in the biosynthesis, in the organic synthesis of peptides, even quite large ones.
And I really feel that over the coming years, many of the proteins that are now being made or are being tried or are being attempted by DNA technology may be ultimately made by protein synthetic methods.
The technology, for example, of purification has improved enormously. The HPLC, the high performance liquid chromatography techniques, are not only much more specific and discerning than the old ion exchange methods and so on,
but they can be made on a very large scale. I was just visiting the biotechnology offshoot of the Carlsberg Laboratory in Copenhagen last week, and I'm impressed with the way that the large HPLC technology has come out from Millipore Waters in Boston
and is now present in many industrial and university laboratories, large enough to prepare, to take kilos in single runs, kilos of synthetic materials for purification, with great success.
And, of course, we have my own baby, which is affinity chromatography, which has also improved to a point where in many cases one can take a crude mixture of biological material or of synthetic material, pass it through an affinity column, that is to say an immobilized ligand column, which would catch only one type of substance,
and go very often from a great mixture of materials to a single component in one step. So between these methods we have now excellent purification techniques for proteins and polypeptides. The most important, of course, is the development of the synthetic methodology itself.
We have all been using the Merrifield solid phase method mostly, in spite of the fact that I think the purest, I think the purest large polypeptide that's been made synthetically was made entirely by hand, that is to say, not machines.
I'm thinking of Wunsch, who made glucagon, which is 50 amino acids. It took about three years, of course, and he had 27 graduate students crystallizing and so on, but it was very pure when it was finished. But that's too much trouble.
So the new methodology improves solid phase synthesis in the following way. Instead of using the most common protecting group, which is TBOC, tertiary butyloxy carbonyl group, for each for the monomer, and which has to be removed at each step with acid,
the newest likely monomer will be the so-called FMOC amino acids, 9-fluoronyl methoxy carbonyl, which can be removed with alkali, and this means a much more gentle, stepwise addition of monomeric units,
and also it has the advantage of permitting the synthesis of peptides containing tryptophan, because tryptophan is unstable to acid. With FMOC, one can make tryptophan peptides and have no problem.
So I do believe that without further complication, one could at the moment look forward to the synthesis of polypeptides of certainly 50 amino acids, I think, in length, and possibly 100 or so, by purely synthetic and completely automatic methods.
I know both the Applied Biosystems Laboratory in California and Milypore Waters in Boston are at the moment about to release to the public, for large amounts of money, machines that will use FMOC and allow the synthesis of fairly large peptides.
Well, I'd like to suggest another extension of proteins and biosynthesis, protein and organic synthesis, which suggests that we might be able to get even larger fragments.
We have been working with a technology which could be called stitching. By stitching, I mean sewing together two large pieces. It was developed originally by Laskowski and Holmberg in New York somewhere, Buffalo, I think.
And we've used it in our own laboratory with several protein systems with considerable success. It's a very, very interesting situation. I'd like to show you what I have in mind. I might just show you first the protein that we began in studying,
which is a protein called staphylococcal nuclease, a nuclease produced by Staph aureus as an extracellular protein from cultures. And I believe the first word is this thing. Here we have staphylococcal nuclease, 149 amino acids,
one tryptophan here. I should point out a lysine, lysine at 48, 49, and another one up here at 5, 6.
It has no disulfide bonds, so there are no problems with SS formation from thiol groups. And the molecule, if denatured completely and allowed to renature,
folds up in three dimensions with a half time of about 200 milliseconds, very rapid folding. The next slide shows the staph nuclease molecule in a simple folded up diagram
taken from the crystallographic work on the three-dimensional structure. This three-dimensional structure was worked out by Dr. Cotton and his colleagues at MIT some years ago. And you will see here a chain which suddenly becomes an anti-parallel pleated sheet
and helix, another anti-parallel pleated sheet, and two more helices closer to the carboxyl terminus. And perhaps one more slide using a notation developed by Jane Richardson at Duke
which is convenient, showing the helices as little twisted bundles and these anti-parallel pleated sheets as arrows.
Now, if you attack this molecule with trypsin which, of course, splits between lysine and arginine, it splits following lysine and arginine. And if you protect the rest of the molecule by adding calcium in here and thymidine diphosphate,
three prime, five prime, thymidine diphosphate, the ligands, the two ligands, protect the rest of the molecule and keep it in a tight conformation. But you can see here, sticking out into the solution,
a loop which contains lysine, lysine, 48, 49 that I showed you before. When trypsin is added to this, you cleave that lysine, lysine bond and also this 5, 6 lysine, lysine up at the end
and you get two pieces, one fragment from 6 to 49 and one from 50 to 149. The two pieces separately, the large piece and the small piece, have no structure of their own but if you mix them together, in spite of this cleavage,
they combine, they form a folded structure which is completely isomorphous with the native molecule so that the same structure is achieved without that bond. Now the important thing that I want to make, the important point I want to make now is that
if one could make large peptide fragments synthetically, it's possible to stitch them together and regenerate the original protein. In this particular case, if you take these two pieces in the presence of calcium and thiamine diphosphate
and add trypsin, but now not in water but in 90% glycerol, if you decrease the water in the solution and increase the glycerol concentration, the equilibrium of the trypsin reaction is shifted towards synthesis instead of cleanage
and this bond can be reformed almost in 100% yield, goes to completion and you regenerate the molecule in quite good yield and activity. So we have then the possibility of making something quite large by taking advantage of this stitching technique.
I'll show you in a moment, we're trying it on another system now. Using such, the next slide perhaps, here we have another photograph of this trypsin cleavage,
giving this small piece and this large piece. Now as a study in chemical modification by synthetic methods, we proceeded to make quite a large number of derivatives of this smaller fragment
which is 43 amino acids and could replace many of the residues here with substitutions that still permitted full activity when this bond was made.
For example, around this calcium, this calcium atom which is required for activity, is liganded by four carboxyl groups, which is a standard situation with calcium. We found that the substitution of any one of those four carboxyl groups
by transforming glutamic acid to glutamine or aspartic acid to asparagine destroyed the ability to reform the protein and the activity was gone. The polynucleotide chain runs up through this groove in the molecule
and there is an arginine residue ordinarily here in the chain which complexes with a phosphate group. If this arginine is replaced by a citrulline, once again the activity is destroyed. So we found a number of changes that were permitted and a number that were not permitted.
So, in a sense, these are what you might call chemical site-directed mutagenesis not involving DNA. On the other hand, of course, the DNA technology is so powerful that at the moment
this kind of thing can be done much more quickly and I borrowed a slide from Dr. David Shortle at Johns Hopkins which shows what sort of success he has had. Here is the same staphylococcal nuclease molecule. These X's along here represent mutations that he and his colleagues have introduced by random mutagenesis
and then insertion into E. coli which would express each one of these separately. They developed a technique for picking out active and inactive single amino acid mutants.
And he has, I think, out of 149 amino acids, he has something like 94 different varieties. It is a very powerful technique. We have recently begun to study another protein using the same methodology
and I should tell you why. This is a molecule known as thioredoxin. It is a very common molecule in nature, particularly in large amounts in a number of bacteria, salmonella, E. coli, etc.
It is involved in catalyzing disulfide bond formation in the maturation of bacteriophage, the conversion of ribonucleotides to deoxyribonucleotides. It has a number of different functions. But for us in particular, it is a nice protein to work with because it is quite small, you see.
It is only 109 amino acids. And its structure has been worked out quite accurately by Bundin in Sweden. It has a single arginine residue here.
What we have been doing, and I should mention first of all why thioredoxin. Down the hall from me in Hopkins is a man called Dr. Ludwig Brand, who is, I think, quite well known in the field of fluorescence. And he has extremely fine equipment now with laser fluorescence
and the proper computer technology for fishing out the four or five decay half-times and so on and so forth. So what we are starting to do now is to take this molecule,
which contains two tryptophanes, one tryptophane here, one tryptophane here, which of course are the ideal amino acid for people interested in fluoroscopy. And he has so far been doing studies on the native molecule
to get some idea of the half-life decay times for these tryptophanes. Now, since it is a university and we have students who have to learn as much as they can, it is a great opportunity to combine some protein chemistry and some genetics.
So in my laboratory at the moment, three students are working on the site-directed immunogenesis of this protein, using the standard cDNA plasmids, lambda, phage, vector, etc.,
which I do not know much about but I am learning. They have made one mutant containing phenylalanine replacing tryptophane here and another one with phenylalanine here. And Lenny Brannan assures me that by studying these he can learn a great deal about the conformation in that region.
I, on the other hand, am anxious to expose these students to some organic chemistry, some peptide chemistry, so what we have done is to block all of the lysine residues along the chain
with citric corneal groups, which can be added very easily, leaving only the arginine residue as the residue sensitive to trypsin. The molecule can then be treated with trypsin and split so that we get this piece and this large piece.
Then the citric corneal can be taken off very easily. The two pieces can be added together. They have some activity without being joined, but at this particular moment we have synthesized this fragment using TBOC,
and we are going to repeat it now using FMOC in the native form, and also have begun some studies on the introduction of various fluorescent amino acids in place of some of these C-terminal ones that are sticking out here,
hoping to be able to get not only additional fluorescent centers, but also centers for energy, the study of energy transfer between the tryptophanes here and something in the C-terminus. So as a teaching situation, it's quite nice. I think that whether they like it or not, they have to stop making mutants and synthesize the peptide.
So it's a good training situation. Well, all in all, let me just finish this business. I really think that with this stitching technique and a little careful designing,
and with the improved synthetic technology, that we really should be able to make proteins certainly this size, I think, one way or another in the next two years. I think this can be developed. It will never equal the DNA technology for very large proteins, of course. But I think in terms of medically important substances,
the many, many, many peptides that are now being used and discovered in medicine, that synthesis will become the important one. I mentioned Copenhagen before. I visited this Carlsberg Biotech Company last week, which is entirely devoted to this kind of thing.
They are concentrating entirely on organic synthesis and have a number of peptides that they have made in rather large quantities
and very clean because of this large-scale HPLC method. They are using not just trypsin and carboxypeptidase and a few other things, but they are using a whole variety of enzymes, including caffeine and other pancreatic proteins, calmatrypsin and so on,
and arranging the C-terminus of the portion to which the smaller piece should be added with different blocking groups that are more specific for the enzyme in question. I think they are doing quite well and are starting to sell some clean things that went through the FDA.
Finally, I'd like to say a word about another area that I really think will become very important in medical science, in industrial medical science for that matter, and that is the whole idea of vaccines, synthetic vaccines.
One is interested in whether or not small pieces of proteins can fold into the three-dimensional structure
that's characteristic of that piece when it's part of the protein molecule. For example, with staph nuclei is the one I showed you before. You have this pleated sheet area and three alpha helical portions.
You can chop the molecule in different ways. The one I'll mention now specifically is the smallest piece, it's 99 to 149, which is prepared with cyanogen bromide cleavage of a methionine residue.
You ask yourself, will such a piece, in solution by itself, take on some three-dimensional structure that resembles what it used to have when it was part of the protein? Now, putting it in immunological terms, if this is an antigenic determinant,
if you make antibodies against nuclease, and this happens to be one of the determinants against which an antibody is made, can you use such a little piece as a competitor or a recognition molecule for the antibody?
Will it remember what it used to look like? So, what we have done with staph nuclei is to simply begin on this kind of, studying this question, is to look at this C-terminal portion running from 99 to 149.
This would be the random form without structure. And we ask the question, is this piece of nuclease in equilibrium with a structure that resembles the native conformation?
If this were an antigenic determinant in the native molecule, then this piece would be recognized by the proper antibody. So, to test that, we made antibodies against native nuclease by injection into rabbits,
and produced, of course, a great mixture of anti-nuclease antibodies, one antibody against each antigenic site on the surface of the globular protein. To clean up the system a little bit, we attached,
we took sepharose and attached to it some nuclease and caught the total antibody population on that column of nuclease sepharose. Then that peak, which was eluted with acid,
was then passed through another column to which had been attached the peptide 99 to 149, and of this whole large population of antibodies, a much smaller population was caught, namely anti-99 to 149. However, this antibody preparation here turned out to be polyvalent.
That is to say, if we added this to nuclease, we got a precipitate, so it had more than one antigenic site. So, we simply took this peak, put it through still another column,
containing only 99 to 126, the peptide attached to the column, and then we got out this one. I'm sorry. We attached to the column 127 to 149 and caught the antibody that we did not want and got anti-99 to 126, which is a non-precipitating antibody.
It does, however, inhibit, it binds to the protein and inhibits the activity, destroys the activity. So we had then a specific antibody that would bind and inactivate nuclease.
By doing some kinetic measurements on the activity of nuclease against DNA, its substrate, with increasing the amounts of anti-99 to 126, we could calculate that that piece, that piece of protein 99 to 126,
existed in a form sufficiently similar to the native structure to bind to the antibody about 1% of the time. It's not a lot, but at least about 1% of the time it looked like it should,
and that's good enough for this kind of synthetic vaccine work that I want to mention. And then we've done this with other bits of proteins, and it turns out that you can't isolate a single determinant, which can be used for the purpose of vaccine preparation.
This was an old experiment with lysozyme many years ago, where we took the loop, there's a loop of amino acid sequence that sticks out from lysozyme, which could be synthesized and attached to a large carrier molecule, injected into rabbits,
and that synthetic mixture produced an antibody that would inactivate lysozyme. So it was like a vaccine against lysozyme, essentially. More reasonably is this situation, which was work done by Ruth Arnone and her colleagues at the Weissmann Institute,
where the coat protein of MS2 virus, it's a coliphase, this is the monomer of the coat protein, could be broken up into three pieces, turned out that this second piece was the antigenically active piece,
and this could be synthesized, attached to a carrier, and could neutralize MS2 virus like any normal vaccine. This principle, I think, is being used now quite widely. I know it, certainly, at the Scripps Institute in California,
at the Weissmann Institute, and a number of other places, people are looking into making pieces of coat proteins, it's mostly viral coat proteins, that are large enough to form a bit of structure that's recognized by the antibody against the total virus.
I know that work is actively going on with hoof and mouth disease, influenza, cholera, and a few other things, and it's based entirely on this idea of the ability of small peptides to remember what they once looked like,
and I think it may become an important aspect of vaccine production. So perhaps I won't talk about nuclear weapons and human rights after all of this stuff.