1967 (V) – Dr. Oliver Smithies

Oliver Smithies:

This is book V beginning March 31^st, 1967 and ending on August of the same year. And it begins with a continuation of membrane cultures, etc. And more attempts at culturing cell. Images again of the thymus. And [Mikhail Levan?] was my technician at that time and I have here an entry that says that ML60, [Mikhail Levan?] who was an enjoyable technician. [00:01:00] And it looks as if I’m drawing to the end on this wild goose chase (inaudible) gamma globulin on April 5^th. A very weird image. [Sub?] agar test the week of April 20^th. [00:02:00] Thymus 24 hours. [Mikhail Levan?] 63 again, [Mikhail Levan?], again. Agar test, etc. Thymus pieces. Wednesday. Oh, sorry, the week of May 1^st, page 23, decided to try to convert myeloma cell that are nonproducers to antibody-producing. Obtained from Ian. That must have been my postdoc Ian. So where were we? Somewhere I got (inaudible) page 23. Decided to convert myeloma cells, etc. [00:03:00] Ian. The setup came from Ian. That’s Ian Walker who was from Australia and worked with me as a postdoc for some time. As I remember we enjoyed each other but didn’t ever produce much together.

So I obtained from him [C1/37 C3H?] now nonproducer. And [C1-158?], a nonproducer from [Mel Cohen?] and a BALB/c nonproducer. [00:04:00] And two BALB/c. And also a BALB/c producer to be cloned for positive cell. How the experiment was going to work is not clear. But here are images of them on the following page with the myeloma cells which I still remember as being amazed at how rapidly they were dividing. How many mitoses there were in the — no, I think I’m wrong. That was a melanoma, not a myeloma. [00:05:00] But there were cells dividing. Which images were from [Mikhail’s?] experiment. And these different cultures. Trying to find single cells, etc. And discover what’s happening. Very small amount of material and try to look at [00:06:00] cultures of cells that might be dividing.

For example on page 39, Thursday, May 18^th, it’s cloning. Since singles could not be found easily yesterday after isolation decided to try some different ways of trying to look at the cell, realizing that when the tiny bubble of cells was present in a dish it couldn’t stabilize it because of loss of water, etc. Three different procedures were tried.

Four were tried with one found. One method. [00:07:00] And this comment. The cells stay fluorescent for several minutes. Trying to culture these antibody-producing cells with May 22^nd, Monday cloning ideas. Plate and allow them to attach at room temperature with carbon dioxide and water. Then afterwards inspect them. [00:08:00] With a comment that [Mikhail?] found it works well.

And back to chemistry again. On Friday, July 27^th, page 51 dinitrobenzene derivative. Fluorodinitrobenzene, etc. Onto a batch of gamma globulin. [00:09:00] Didn’t work because the next page, page 53, says the procedure may have failed at several stages. And so set up various things. N-terminal blockade considered on Wednesday, August 2^nd. Back to chemistry on labeling N-terminal sequences.

This is in 1967. So that picking this up again Wednesday, August 2^nd, try to label proteins with dinitrofluorobenzene on the N terminus. [00:10:00] Continuing on the following pages with blockade of the N-terminal peptide. More division again. Rather unclear. I’ve been talking to Gordon Dixon about it. As evident on page 61. Gordon Dixon. G.H. Dixon suggests arginine blockade, etc., etc. But why I was doing the experiment is far from clear.

[00:11:00] Thursday, August 3^rd, some clues as to what they were being used for because it says that decided to make a larger quantity of dinitrobenzene acetyl and succinyl derivatives, that’s N-terminal derivatives, and of their peptides for better column development.

So here is the arginine blockade that Gordon suggested on Friday, August 4^th. Succinylated the gamma globulin. Already made on page 63. [00:12:00] Treated with trypsin. Carboxypeptidase B and blockade Saturday, August 5^th. For unclear reasons. Continuing with these experiments. [00:13:00] [00:14:00] It was obvious I wanted to blockade carboxypeptidase B for some reason but why I was attempting these peptides is far from clear from the notebooks. [00:15:00] Some quite pretty images of gels on page 88 and 89 with the effect of trypsin and carboxypeptidase and fluorescein, fluorescent material, etc. The conclusion is that trypsin acted OK and that carboxypeptidase query contaminated also but that the relevant peptides are — I think insoluble is the meaning of the word. It says I soluble. Because the next page is concerned with [00:16:00] solubilization of the dinitrobenzene peptides. Still effectively insoluble. And urea is very troublesome for the reagents.

Many, many images on pages 98 and 99 of these various peptidase and tryptic digests with or without mercaptoethanol. So preparation of the N-terminal prospect. [00:17:00] Trying the fingerprints again. Monday, August 13^th, page 105. Necessary to establish the need or not to cleave the disulfide bonds for fingerprinting of tryptic peptide.

And here are some fingerprints on page 111. [00:18:00] Not very satisfactory by look of them. Still these are Bence-Jones proteins because on page 115 I’m talking about one milligram per 4 ml of [Dylan?] and [Eddie?], which were Bence-Jones proteins. And succinyl gamma globulin, etc.

[00:19:00] Rough test of carboxypeptidases on Tuesday, August 15^th. These experiments I think must have been driven by my interest in antibody variability but it’s far from clear from the notebook. [00:20:00] I see myself getting into a trap that eventually was a problem of isolating light chains to try to see what was happening in antibody variability. Friday, August 18^th, bulk light chains. Decided to isolate bulk chains to try to see peptides better and compare [00:21:00] gamma globulin and L and BJ fingerprints. That probably means light chains and Bence-Jones protein fingerprints.

Using the two proteins as I had plenty of supply of [Eddie?] and [Dylan?]. Looking at the fingerprints. Conclusion is these whole gamma globulin fingerprints are too complicated. Too complex. Purify the light chains for this phase of the work. Meanwhile do exhaustive acetylation and succinylation fingerprints on the Bence-Jones protein. [00:22:00] Reduce and alkylate it also. Get carboxypeptidase in good shape and return to the direct approach using Bence-Jones as models first. So I tried to make bulk light chains on page 131. Needing to try to purify the carboxypeptidase.

[00:23:00] So isolating light chains on Tuesday, August 22^nd. [00:24:00] Stop it, Jenny, please. [00:25:00] Still trying to separate light chains on Friday, August 25^th. [00:26:00] Almost the last entry in this book on page 153. Excellent separation of light chain and heavy chain on this column. Which was I think not very clear what the column was. [00:27:00] As on page 153 I said the excellent separation. I can assume that the final volume contains about 50 milligrams of light chains. The last entry in this book shows some traces. Some separation with portion kept as heavy chain and a portion concentrated rerun for the light chain. And pooled V155 light chain reasonably separated from heavy chain [00:28:00] on the second run. So it says here then that on Monday reran. Obtained in good shape. That is obtained the light chains in good shape. End of this book V.