Separatome-based protein expression and purification platform

Patent metadata

Field	Value
Publication number	US-10030257-B2
Application number	US-201715428220-A
Country	US
Kind code	B2
Filing date	Feb 9, 2017
Priority date	Mar 13, 2012
Publication date	Jul 24, 2018
Grant date	Jul 24, 2018

Abstract

Provided is a separatome-based recombinant peptide, polypeptide, and protein expression and purification platform based on the juxtaposition of the binding properties of host cell genomic peptides, polypeptides, and proteins with the characteristics and location of the corresponding genes on the host cell chromosome, such as that of E. coli, yeast, Bacillus subtilis or other prokaryotes, insect cells, mammalian cells, etc. The separatome-based protein expression and purification platform quantitatively describes and identifies priority deletions, modifications, or inhibitions of certain gene products to increase chromatographic separation efficiency, defined as an increase in column capacity, column selectivity, or both. Moreover, the separatome-based protein expression and purification platform provides a computerized knowledge tool that, given separatome data and a target recombinant peptide, polypeptide, or protein, intuitively suggests strategies leading to efficient product purification. The separatome-based protein expression and purification platform is an efficient bioseparation system that intertwines host cell expression systems and chromatography.

First claim

What is claimed is:

1. A method of purifying a target peptide, polypeptide, or protein, comprising the steps of:
1) recombinantly or non-recombinantly expressing said target peptide, polypeptide, or protein in a host cell wherein upon chromatographic purification, the chromatographic separation efficiency of said expressed target peptide, polypeptide, or protein is improved in an amount in the range of from about 3% to about 50%,
wherein the genome of said host cell:
a) is a reduced genome,
b) a modified genome, or
c) a genome in which expression of genes is reduced or completely inhibited,

wherein genes that are deleted, modified, or the expression of which is reduced or completely inhibited in said host cell code for peptides, polypeptides, or proteins that impair the chromatographic separation efficiency of said expressed target peptide, polypeptide, or protein,
wherein deletion, modification, reduction of expression, or complete inhibition of expression of said genes in said host cell improves the chromatographic separation efficiency of said expressed target peptide, polypeptide, or protein in an amount in the range of from about 3% to about 50% compared to the chromatographic separation efficiency of said expressed target peptide, polypeptide, or protein when said genes are not deleted, not modified, or the expression of which is not reduced or completely inhibited in said host cell, respectively, and
wherein genes that are deleted, modified, or the expression of which is reduced or completely inhibited in the genome of said host cell are identified, scored, and ranked according to chromatographic relevance in adversely affecting said chromatographic separation efficiency of said expressed target peptide, polypeptide, or protein by:
i) equilibrating an affinity chromatography column comprising an affinity ligand bound to a solid phase, or an adsorption-based, non-affinity chromatography column comprising an adsorption-based, non-affinity chromatography medium, using a mobile loading or eluting phase, or an operational variable;
ii) in the case where said expressed target peptide, polypeptide, or protein is not secreted from said host cell, fractionating a lysate of said host cell, or
in the case where said expressed target peptide, polypeptide, or protein is secreted from said host cell, fractionating the culture medium in which said host cell is grown,
on either of said columns by applying an elution gradient to elute host cell peptide, polypeptide, or protein fractions from said column;
iii) identifying, quantifying, scoring, and ranking host cell peptides, polypeptides, or proteins in fractions eluted from either of said columns, respectively;
iv) assessing the metabolic role of said host cell peptides, polypeptides, or proteins identified in step iii) that affect column capacity; and
v) reducing or modifying the genome of said host cell, or reducing or completely inhibiting expression of genes in said host cell, thereby modifying the proteome of said host cell and increasing the chromatographic separation efficiency of said expressed target peptide, polypeptide, or protein, based on steps iii) and iv) in an amount in the range of from about 3% to about 50% compared to the chromatographic separation efficiency of said expressed target peptide, polypeptide, or protein when said genes are not deleted, not modified, or the expression of which is not inhibited in said host cell, respectively,
wherein host cell peptides, polypeptides, and proteins are scored, and ranked in order of importance, in step iii) according to the following equation:

importance
i

=

∑
j

⁢

[

b
1

⁡

(

y

c
j

y
max

)

⁢

(

h

i
⁢

,
j

h

i
,
total

)

⁢

(

h

i
⁢

,
j

h

j
,
total

)

⁢

(

MW
i

MW
ref

)

α

]

i

wherein b 1 =scaling parameter; y cj and y max =concentration of mobile phase eluent in fraction (j) and maximum value, respectively; h i,j and h i,total =the amount of protein (i) in fraction (j) and total bound protein (i), respectively; h j,total =total amount of protein in fraction (j); MW i =molecular weight of protein (i); MW ref =molecular weight of a reference protein within the separatome; a=steric factor; and i=protein,
wherein:
ratios of y's and h's adopt values between 0 and 1;
a protein that remains bound and requires stringent conditions for elution exhibits a y ratio

(

y

c
j

y
max

)

 close

Assignees

• Univ Arkansas
• Univ Of Pittsburgh—Of The Commonwealth System Of Higher Education

Inventors

• Brune Ellen M
• Beitle Jr Robert R
• Ataai Mohammad M
• Bartlow Patrick R
• Henry Ralph L

Classifications

• C12N15/74

  Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora · CPC title

• B01D15/203

  Equilibration or regeneration · CPC title

• B01D15/362

  Cation-exchange · CPC title

• C12N1/20

  Bacteria; Culture media therefor · CPC title

• C12P21/00 Primary

  Preparation of peptides or proteins (single cell protein C12N1/00) · CPC title