# US 10,696,963 B2 · Selective optimization of a ribosome binding site for protein production

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- Number: US 10,696,963 B2
- Document ID: US10696963B2
- Status: Granted
- Kind: B2
- Application number: 15/535,686
- Filed: 2015-12-15
- Published: 2018-09-27
- Granted: 2020-06-30
- Earliest claimed: 2014-12-16
- Assignees: Cloneopt AB
- Applicants: CloneOpt AB
- Inventors: Daniel Daley; Kiavash Mirzadeh; Stephen Toddo; Suchithra Guntur
- Web page: https://patentgrep.com/patent/US10696963B2

## CPC

- C12N 15/1086: {Preparation or screening of expression libraries, e.g. reporter assays}
- C12N 15/67: General methods for enhancing the expression
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

A method for optimising a nucleotide sequence for protein expression in a host cell, comprising the steps of: (a) constructing an expression library comprising a large number of variants of the sequence to be expressed operatively cloned in an expression vector, wherein (i) the sequence of the 6 nucleotides immediately upstream (5′-direction) of the first codon of the coding sequence to be expressed is completely or partially randomized; (ii) the sequence of the second and third codons of the coding sequence to be expressed is randomized, wherein the randomization of the second and third codons is limited to changes not altering the amino-acids encoded by said codons; (b) screening the library with regard to efficiency of protein expression in the host cell; and (c) selecting a sequence resulting in a desired level of efficiency of protein expression.

## Claims

### Claim 1

A method for optimising a nucleotide sequence for protein expression in a host cell comprising:
constructing an expression library comprising a plurality of variants of a coding sequence to be expressed, wherein individual members of the plurality are operatively cloned in an expression vector, wherein
i. a sequence of 6 nucleotides immediately upstream in the 5′ direction of the sequence of a first codon of the coding sequence to be expressed is completely randomized;
ii. sequences of a second codon and a third codon of the coding sequence to be expressed are randomized, wherein the randomization of the second and third codons results in changes that do not alter the amino-acids encoded by said second and third codons;
screening the expression library to determine efficiency of protein expression in the host cell; and
selecting an optimized nucleotide sequence that results in a desired level of efficiency of protein expression.

### Claim 2 (depends on 1)

The method according to claim 1, wherein constructing the expression library comprises conducting a PCR reaction using a set of degenerate primers comprising:
(i) a sequence of least 6 completely randomised nucleotides immediately upstream in the 5′-direction of the first codon of the coding sequence to be expressed;
(ii) the sequence encoding the first codon of the coding sequence to be expressed; and
(iii) a randomized sequence of the second and third codons of the coding sequence to be expressed, wherein the randomization of the sequence of the second and third codons results in changes that do not alter the amino-acids encoded by said second and third codons;
or the complement of said sequences in (i)-(iii).

### Claim 3 (depends on 1)

The method according to claim 1, wherein the library comprises at least 1000 variants of the 6 nucleotides immediately upstream in the 5′-direction of the first codon of the sequence to be expressed.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the library comprises at least 2 variants of the sequence of the second and third codons.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the library comprises at least 4000 variants of the nucleotide sequence to be optimised.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the operative cloning in the expression vector results, when the expression vector is introduced into a suitable host cell under suitable conditions, in expression of an mRNA molecule comprising at least the following features, in the following order from the 5′ direction: (i) the sequence of the 6 nucleotides immediately upstream in the 5′-direction of the first codon of the coding sequence to be expressed and (ii) the coding sequence to be expressed.

### Claim 7 (depends on 1)

The method according to claim 1, wherein the operative cloning in the expression vector results, when the expression vector is introduced into a suitable host cell under suitable conditions, in expression of an mRNA molecule comprising at least the following features, in the following order from the 5′ direction: (i) a Shine-Dalgarno sequence; (ii) the sequence of the 6 nucleotides immediately upstream in the 5′-direction of the first codon of the coding sequence to be expressed; and (iii) the coding sequence to be expressed.

### Claim 8 (depends on 1)

The method according to claim 1, wherein the library is constructed such that the sequence to be expressed is expressed as a fusion protein with a detectable marker.

### Claim 9 (depends on 8)

The method according to claim 8, wherein the detectable marker is a fluorescent protein.

### Claim 10 (depends on 9)

The method according to claim 9, wherein the screening is performed by flow cytometry.

### Claim 11 (depends on 1)

The method according to claim 1, wherein the expression vector comprises a Shine-Dalgarno sequence.

### Claim 12 (depends on 1)

The method according to claim 1, wherein the expression vector is a high copy number vector.

### Claim 13 (depends on 1)

The method according to claim 1, wherein the host cell is a prokaryotic cell.

### Claim 14 (depends on 1)

The method according to claim 1, wherein the host cell is Escherichia coli.

### Claim 15 (depends on 1)

The method according to claim 1, wherein the expression vector comprises a T7 promoter, a tac promoter, or a lac promoter.

### Claim 16 (depends on 2)

The method according to claim 2, wherein the library comprises at least 1000 variants of the 6 nucleotides immediately upstream in the 5′-direction of the first codon of the sequence to be expressed.

### Claim 17 (depends on 2)

The method according to claim 2, wherein the library comprises at least 2 variants of the sequence of the second and third codons.

### Claim 18 (depends on 2)

The method according to claim 2, wherein the library comprises at least 4000 variants of the nucleotide sequence to be optimised.

### Claim 19 (depends on 2)

The method according to claim 2, wherein the operative cloning in the expression vector results, when the expression vector is introduced into a suitable host cell under suitable conditions, in expression of an mRNA molecule comprising at least the following features, in the following order from the 5′ direction: (i) the sequence of the 6 nucleotides immediately upstream in the 5′-direction of the first codon of the coding sequence to be expressed and (ii) the coding sequence to be expressed.

### Claim 20 (depends on 2)

The method according to claim 2, wherein the operative cloning in the expression vector results, when the expression vector is introduced into a suitable host cell under suitable conditions, in expression of an mRNA molecule comprising at least the following features, in the following order from the 5′ direction: (i) a Shine-Dalgarno sequence; (ii) the sequence of the 6 nucleotides immediately upstream in the 5′-direction of the first codon of the coding sequence to be expressed; and (iii) the coding sequence to be expressed.

### Claim 21 (depends on 2)

The method according to claim 2, wherein the library is constructed such that the sequence to be expressed is expressed as a fusion protein with a detectable marker.

### Claim 22 (depends on 21)

The method according to claim 21, wherein the detectable marker is a fluorescent protein.

### Claim 23 (depends on 16)

The method according to claim 16, wherein the library is designed to comprise at least 2 variants of the sequence of the second and third codons.

### Claim 24 (depends on 16)

The method according to claim 16, wherein the library comprises at least 4000 variants of the nucleotide sequence to be optimised.

### Claim 25 (depends on 23)

The method according to claim 23, wherein the library comprises at least 4000 variants of the nucleotide sequence to be optimised.

### Claim 26 (depends on 3)

The method according to claim 3, wherein the library comprises at least 2 variants of the sequence of the second and third codons.

### Claim 27 (depends on 3)

The method according to claim 3, wherein the library comprises at least 4000 variants of the nucleotide sequence to be optimised.

### Claim 28 (depends on 27)

The method according to claim 27, wherein the library comprises at least 4000 variants of the nucleotide sequence to be optimised.

### Claim 29 (depends on 28)

The method according to claim 28, wherein the library is constructed such that the sequence to be expressed is expressed as a fusion protein with a detectable marker.

### Claim 30 (depends on 29)

The method according to claim 29, wherein the detectable marker is a fluorescent protein.