Positional control of selenium insertion in polypeptides for X-ray crystallography

• US 5,700,660 A
• Filed: 06/07/1995
• Issued: 12/23/1997
• Est. Priority Date: 05/24/1993
• Status: Expired due to Term

• Current Assignee: University Of Massachusetts
• Original Assignee: University Of Massachusetts
• Inventors: Leonard, Jack L., Newburger, Peter E.
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): Longton, Enrique D.
• Application Number:
  US08/473,496
• Time in Patent Office: 930 Days
• Field of Search: 435/69.1, 378/73, 530/400
• US Class Current: 435/69.1
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: 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
• CPC Class Codes: C12N 15/79 Vectors or expression systems specially adapted for eukaryotic hosts
• CPC Class Codes: C12N 9/0065 acting on hydrogen peroxide as acceptor (1.11)
• CPC Class Codes: C12P 21/00 Preparation of peptides or proteins single cell protein C12N1/00
• CPC Class Codes: 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

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

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.

2. 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.

3. 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.

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

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

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

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

8. 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.

9. 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'"'"'-A₂₁ U₂₂ G₂₃ R₂₄ G₂₅ -3'"'"' (SEQ ID NO:

2) and form a first loop,

d) nucleotides 60 to 68 are 5'"'"'-Y₆₀ R₆₁ N₆₂ N₆₃ N₆₄ -N₆₅ U₆₆ A₆₇ V₆₈ -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'"'"'-A₃₈ R₃₉ A₄₀ N₄₁ N₄₂ N₄₃ N₄₄ N₄₅ N₄₆ N₄₇ N₄₈ -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.

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

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

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

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

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

5).

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

6).

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