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CN-112480218-B - Method for assembling proteins having multiple subunits

CN112480218BCN 112480218 BCN112480218 BCN 112480218BCN-112480218-B

Abstract

The present disclosure provides a method for assembling a protein having a plurality of subunits, the method comprising (a) providing a plurality of first subunits, (b) providing a plurality of second subunits, wherein the second subunits are modified relative to the first subunits, (c) contacting the first subunits with the second subunits at a first ratio to form a plurality of proteins having the first and second subunits, wherein the plurality of proteins have a plurality of ratios of the first and second subunits, and (d) fractionating the plurality of proteins to enrich the protein having a second ratio of the first and second subunits, wherein the second ratio is one second subunit per (n-1) first subunit, wherein 'n' is the number of subunits comprising the protein.

Inventors

  • R. DAVIS
  • R.CHEN
  • BIBILLO ARKADIUSZ
  • D. Cologne Boulogne
  • M. Duoerwate

Assignees

  • 吉尼亚科技公司

Dates

Publication Date
20260505
Application Date
20131107
Priority Date
20121109

Claims (13)

  1. 1. A method for assembling a nanopore having a plurality of subunits, wherein the method comprises: (a) Providing a plurality of first subunits; (b) Providing a plurality of second subunits, wherein the second subunits are modified relative to the first subunits; (c) Contacting the first subunit with the second subunit at a first ratio to form a plurality of nanopores having the first subunit and the second subunit, wherein the plurality of nanopores have a plurality of ratios of the first subunit to the second subunit, and (D) Fractionating the plurality of nanopores using ion exchange chromatography to enrich the nanopores having a second ratio of the first subunit to the second subunit, wherein the second ratio is 1 second subunit per 6 first subunits; wherein the first subunit is wild-type or recombinant alpha-hemolysin; wherein the second subunit is recombinant alpha-hemolysin; Wherein the second subunit comprises a purification tag, and wherein the method further comprises (e) performing a reaction to attach a polymerase to the purification tag using non-covalent interactions.
  2. 2. A method for assembling a nanopore having a plurality of subunits, the method comprising: (a) Providing a plurality of first subunits; (b) Providing a plurality of second subunits, wherein the second subunits are modified relative to the first subunits; (c) Contacting the first subunit with the second subunit at a first ratio to form a plurality of nanopores having the first subunit and the second subunit, wherein the plurality of nanopores have a plurality of ratios of the first subunit to the second subunit, and (D) Fractionating the plurality of nanopores using ion exchange chromatography to enrich the nanopores with a second ratio of 2 second subunits per 5 first subunits and a single polymerase attached to each of the second subunits; wherein the first subunit is wild-type or recombinant alpha-hemolysin; wherein the second subunit is recombinant alpha-hemolysin; Wherein the second subunit comprises a purification tag, and wherein the method further comprises (e) performing a reaction to attach a polymerase to the purification tag using non-covalent interactions.
  3. 3. The method of any one of claims 1-2, wherein the nanopore is at least 80% homologous to alpha-hemolysin.
  4. 4. The method of any one of claims 1-2, wherein the first subunit comprises a polyhistidine tag.
  5. 5. The method of any one of claims 1-2, wherein the first subunit is wild-type alpha-hemolysin.
  6. 6. The method of any one of claims 1-2, wherein the first and second subunits are recombinant alpha-hemolysin.
  7. 7. The method of any one of claims 1-2, wherein the first ratio is equal to the second ratio.
  8. 8. The method of any one of claims 1-2, wherein the first ratio is greater than the second ratio.
  9. 9. The method of any one of claims 1-2, further comprising inserting a nanopore having the second ratio of subunits into a lipid bilayer.
  10. 10. A method of sequencing a nucleic acid molecule by means of a nanopore assembled by the method of any of claims 1-2, comprising sequencing a nucleic acid molecule by means of a nanopore having the second ratio subunit.
  11. 11. A nanopore comprising a plurality of first and second subunits, wherein the ratio between the first and second subunits is 1 second subunit per 6 first subunits or 2 second subunits per 5 first subunits; Wherein the first subunit is wild-type or recombinant alpha-hemolysin, wherein the second subunit is recombinant alpha-hemolysin; Wherein the second subunit comprises a purification tag, and wherein the polymerase is non-covalently attached to the purification tag.
  12. 12. The nanopore according to claim 11, wherein the nanopore is at least 80% homologous to a-hemolysin.
  13. 13. The nanopore of claim 11, wherein the first subunit comprises a polyhistidine tag.

Description

Method for assembling proteins having multiple subunits The application is a divisional application of an application patent application with the name of 'sequencing by using labeled nucleic acid', the application date is 2013, 11, 07, and the application number is 201380058224.1. Cross reference The present application claims the benefit of U.S. provisional patent application serial No. 61/724,869 filed 11/9/2012, U.S. provisional patent application serial No. 61/737,621 filed 12/14/2012, and U.S. provisional patent application serial No. 61/880,407 filed 9/2013, each of which is incorporated herein by reference in its entirety. Technical Field The present invention relates to the field of biotechnology, more specifically to a method for assembling proteins having multiple subunits. Background Nucleic acid sequencing is a process that can be used to provide sequence information for a nucleic acid sample. Such sequence information may be useful in diagnosing and/or treating a subject. For example, the nucleic acid sequences of a subject can be used to identify, diagnose, and potentially develop therapies for genetic diseases. As another example, research into pathogens may lead to treatment for infectious diseases. There are available methods for sequencing nucleic acids. However, such methods are expensive and may not provide sequence information for a certain period of time and with the accuracy that may be required for diagnosing and/or treating a subject. Disclosure of Invention Methods of nucleic acid sequencing single stranded nucleic acid molecules through a nanopore may have a sensitivity that may be insufficient or inadequate to provide a date for diagnostic and/or therapeutic purposes. The nucleobases (e.g., adenine (a), cytosine (C), guanine (G), thymine (T), and/or uracil (U)) that make up the nucleic acid molecule may not provide signals that are significantly different from one another. In particular, purines (i.e., a and G) have similar sizes, shapes, and charges to one another and in some cases provide signals that are not significantly different. In addition, the pyrimidines (i.e., C, T and U) are of similar size, shape, and charge to each other and in some cases provide a non-distinct signal. There is recognized herein a need for improved methods for nucleic acid molecule recognition and nucleic acid sequencing. In some embodiments, a nucleotide incorporation event (e.g., incorporation of a nucleotide into a nucleic acid strand complementary to a template strand) labels the nanometer Kong Chengshi and/or releases the label from the nucleotide detected through the nanopore. The incorporated bases (i.e., A, C, G, T or U) can be identified because a unique tag is released and/or presented for each type of nucleotide (i.e., A, C, G, T or U). In some embodiments, the label is attributed to successfully incorporated nucleotide based on the period of time that the label is detected to interact with the nanopore. The period of time may be longer than the period of time associated with the free flow of the nucleotide label through the nanopore. The detection period of successfully incorporated nucleotide labels may also be longer than the period of unincorporated nucleotides (e.g., nucleotides mismatched with the template strand). In some cases, the polymerase is attached to the nanopore with the nanoparticle Kong Dige (e.g., covalently attached) and the polymerase performs a nucleotide incorporation event. When the labeled nucleotide is associated with the polymerase, the label can be detected through the nanopore. In some cases, unincorporated labeled nucleotides pass through the nanopore. The method can distinguish between labels associated with unincorporated nucleotides and labels associated with incorporated nucleotides based on the length of time that the labeled nucleotide is detected through the nanopore. In one embodiment, unincorporated nucleotides are detected in less than about 1 millisecond through the nanopore and incorporated nucleotides are detected in at least about 1 millisecond through the nanopore. In some embodiments, the polymerase has a slow kinetic step in which the label is detectable through the nanopore in at least 1 millisecond with an average detection time of about 100 ms. The polymerase may be a mutant phi29 DNA polymerase. The polymerase can be mutated to reduce the rate at which the polymerase incorporates nucleotides into a nucleic acid strand (e.g., a growing nucleic acid strand). In some cases, the rate of incorporation of a nucleotide into a nucleic acid strand can be reduced by functionalizing the nucleotide and/or the template strand to provide steric hindrance (such as, for example, by methylation of the template nucleic acid strand). In some cases, the rate is reduced by incorporating methylated nucleotides. In one aspect, a method of sequencing a nucleic acid sample with the aid of a nanopore in a membrane includes (a) providing labeled nucleotides to a re