# US 5,700,660 A · Positional control of selenium insertion in polypeptides for X-ray crystallography

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- Number: US 5,700,660 A
- Document ID: US5700660A
- Status: Granted
- Kind: A
- Application number: 08/473,496
- Filed: 1995-06-07
- Granted: 1997-12-23
- Earliest claimed: 1993-05-24
- Assignees: University of Masachusetts Medical Center
- Inventors: Jack L. Leonard; Peter E. Newburger
- Web page: https://patentgrep.com/patent/US5700660A

## CPC

- C12N 9/0065: {acting on hydrogen peroxide as acceptor (1.11)}
- A01K 67/0275: Genetically modified vertebrates, e.g. transgenic
- C12N 15/10: Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34)
- C12N 15/79: Vectors or expression systems specially adapted for eukaryotic hosts
- C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00)
- G01N 23/20: by using diffraction of the radiation by the materials, e.g. for investigating crystal structure; by using scattering of the radiation by the materials, e.g. for investigating non-crystalline materials; by using reflection of the radiation by the materials
- A01K 2217/05: Animals comprising random inserted nucleic acids (transgenic)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The invention provides methods for determining the structure of a polypeptide by a) transfecting a cell with (i) a first nucleic acid encoding the polypeptide, wherein at least one, specific codon of mRNA transcribed from the first nucleic acid is replaced by the codon UGA, and (ii) a second nucleic acid, operably linked to the first nucleic acid, that directs the translation of the UGA codon as selenocysteine only when the cell can obtain selenium from the medium in which it is grown; b) growing the cell in selenium-containing growth medium under conditions in which the cell incorporates at least one selenocysteine residue into the polypeptide at a specific location; c) isolating the polypeptide from the cell or the growth medium; d) forming a crystal of the polypeptide; and e) performing X-ray crystallography on the crystal, wherein the selenocysteine residue is used to determine the structure of the polypeptide.

## Claims

### Claim 1

A method for determining the structure of a heterologous polypeptide that does not contain selenocysteine in its native form, said method comprising
(1) transfecting a cell with (i) a first nucleic acid encoding said polypeptide, wherein at least one, specific codon of mRNA transcribed from said first nucleic acid is replaced by the codon UGA, and (ii) a second nucleic acid operably linked to said first nucleic acid, said second nucleic acid directing the translation of said UGA codon as selenocysteine only when said cell can obtain selenium from the medium in which said cell is grown, wherein said second nucleic acid is derived from the 3' untranslated region of a gene encoding mammalian glutathione peroxidase and comprises a continuous stretch of at least 79 nucleotides comprising three stem elements, each having a 5' half and a 3' half, and three loop elements, each having a 5' end and a 3' end,
wherein the stem elements comprise
a) a base stem comprising at least 16 nucleotides that can form 8 complementary pairs of nucleotides,
b) a lower stem comprising at least 16 nucleotides that can form 8 complementary pairs of nucleotides, the first nucleotide of the 5' half of the lower stem being bound to the last nucleotide of the 5' half of the base stem, and the first nucleotide of the 3' half of the lower stem being bound to the last nucleotide of the 3' half of the base stem, and
c) an upper stem comprising at least 22 nucleotides that can form 11 complementary pairs of nucleotides,
wherein the loop elements comprise
d) a first loop consisting of 5'-AUGRG-3' (SEQ ID NO:2), the 5'-A being bound to the last nucleotide of the 5' half of the lower stem and the 3'-G being bound to the first nucleotide of the 5' half of the upper stem,
e) a second loop consisting of 5'-YRNNNNUAV-3' (SEQ ID NO:3), the 5'-Y being bound to the first nucleotide of the 3' half of the upper stem and the 3'-V being bound to the last nucleotide of the 3' half of the lower stem, and
f) a third, apical loop consisting of 5'-ARANNNN-NNNN-3' (SEQ ID NO:4), the 5'-A being bound to the last nucleotide of the 5' half of the upper stem and the 3'-N being bound to the last nucleotide of the 3' half of the upper stem, and wherein each A is adenine, G is quanine, N is adenine, guanine, cytosine, or uracil, R is quanine or adenine, U is uracil, V is any nucleotide except thymidine or uracil, and Y is uracil or cytosine;
(2) growing said cell in selenium-containing growth medium under conditions in which said cell incorporates at least one selenocysteine residue into said polypeptide at a specific location;
(3) isolating said polypeptide from said cell or said growth medium;
(4) forming a crystal of said polypeptide; and
(5) performing X-ray crystallography on said crystal, wherein said selenocysteine residue is used to determine the structure of said heterologous polypeptide.

### Claim 2 (depends on 1)

A method of claim 1, wherein at least two, specific codons of mRNA transcribed from said first nucleic acid are each replaced by the codon UGA, and wherein the cell incorporates at least two selenocysteine residues into said polypeptide at specific locations, at least two of the selenocysteine residues are combined to form a selenocystine residue, and said selenocystine residue is used to determine the structure of said polypeptide in the X-ray crystallography.

### Claim 3 (depends on 1)

A method of claim 1, wherein prior to forming a polypeptide crystal, the selenocysteine residue has been modified to form a selenide, selenoxide, seleninic acid, selenonic acid, selenone, or a seleno-sulfur group, and wherein the modified selenocysteine residue is used to determine the structure of said polypeptide.

### Claim 4 (depends on 1)

A method of claim 1, wherein the X-ray crystallography involves multi-wavelength anomalous diffraction analysis.

### Claim 5 (depends on 1)

A method of claim 1, wherein X-rays used in the X-ray crystallography have wavelengths between about 0.3 to 3.0 Angstroms.

### Claim 6 (depends on 5)

A method of claim 5, wherein said X-rays are produced by synchrotron radiation.

### Claim 7 (depends on 1)

A method of claim 1, wherein said growth medium comprises at least 1 ng of selenium per milliliter.

### Claim 8 (depends on 1)

A method of claim 1, wherein said second nucleic acid further comprises a first mutually exclusive multiple restriction site attached to the first nucleotide of the 5' half of the base stem and a second mutually exclusive multiple restriction site attached to the first nucleotide of the 3' half of the base stem.

### Claim 9 (depends on 1)

A method of claim 1, wherein said second nucleic acid is synthetically derived, and comprises a continuous stretch of 87 nucleotides, wherein
a) nucleotides 1 to 8 are complementary to nucleotides 87 to 80, respectively, and when base-paired together form a base stem consisting of 16 nucleotides in 8 complementary pairs of nucleotides,
b) nucleotides 9 to 20 and 69 to 79 when base-paired together form a lower stem consisting of at least 8 complementary pairs of nucleotides,
c) nucleotides 21 to 25 are 5'-A.sub.21 U.sub.22 G.sub.23 R.sub.24 G.sub.25 -3' (SEQ ID NO:2) and form a first loop,
d) nucleotides 60 to 68 are 5'-Y.sub.60 R.sub.61 N.sub.62 N.sub.63 N.sub.64 -N.sub.65 U.sub.66 A.sub.67 V.sub.68 -3' (SEQ ID NO:3) and form a second loop,
e) nucleotides 26 to 37 and nucleotides 49 to 59 when base-paired together form an upper stem of at least 11 complementary pairs of nucleotides, and
f) nucleotides 38-48 are non-complementary and are 5'-A.sub.38 R.sub.39 A.sub.40 N.sub.41 N.sub.42 N.sub.43 N.sub.44 N.sub.45 N.sub.46 N.sub.47 N.sub.48 -3' (SEQ ID NO:4) and form a third, apical loop, and
wherein A is adenine, G is guanine, N is adenine, guanine, cytosine, or uracil, R is guanine or adenine, U is uracil, V is any nucleotide except thymidine or uracil, and Y is uracil or cytosine.

### Claim 10 (depends on 1)

A method of claim 1, wherein said cell is an insect cell.

### Claim 11 (depends on 1)

A method of claim 1, wherein said cell is a yeast cell.

### Claim 12 (depends on 1)

A method of claim 1, wherein said cell is a mammalian cell.

### Claim 13 (depends on 1)

A method of claim 1, wherein said method is used to detect a binding partner of said polypeptide.

### Claim 14 (depends on 1)

A method of claim 1, wherein said second nucleic acid has the sequence of FIG. 2 (SEQ ID NO:5).

### Claim 15 (depends on 1)

A method of claim 1, wherein said second nucleic acid has the sequence of FIG. 3 (SEQ ID NO:6).

### Claim 16 (depends on 1)

A method of claim 1, wherein at least one of said UGA codons is introduced into said first nucleic acid by site-directed mutagenesis.