US 12,031,168 B2
Methods for controlling protease production
Marja Paloheimo, Rajamäki (FI); Susanna Mäkinen, Rajamäki (FI); Peter Punt, Zeist (NL); Kari Juntunen, Rajamäki (FI); Terhi Puranen, Rajamäki (FI); and Jari Vehmaanperä, Rajamäki (FI)
Assigned to ROAL OY, Rajamäki (FI)
Filed by ROAL OY, Rajamäki (FI)
Filed on Dec. 9, 2020, as Appl. No. 17/116,353.
Application 17/116,353 is a continuation of application No. 15/552,387, granted, now 10,883,131, previously published as PCT/FI2016/050108, filed on Feb. 19, 2016.
Claims priority of application No. 20155112 (FI), filed on Feb. 20, 2015.
Prior Publication US 2021/0102231 A1, Apr. 8, 2021
Int. Cl. C12P 21/02 (2006.01); C07K 14/37 (2006.01); C07K 16/14 (2006.01); C12N 9/02 (2006.01); C12N 9/24 (2006.01); C12N 9/42 (2006.01); C12N 9/48 (2006.01); C12N 15/80 (2006.01)
CPC C12P 21/02 (2013.01) [C07K 14/37 (2013.01); C07K 16/14 (2013.01); C12N 9/0061 (2013.01); C12N 9/2405 (2013.01); C12N 9/2437 (2013.01); C12N 9/48 (2013.01); C12N 15/80 (2013.01); C12Y 110/03002 (2013.01); C12Y 302/01004 (2013.01)] 10 Claims
OG exemplary drawing
 
1. A protein preparation comprising spent culture broth from production of a heterologous recombinant protein of interest in a host cell having at least one inactivated chromosomal gene wherein:
the at least one inactivated chromosomal gene comprises a nucleic acid sequence encoding a polypeptide comprising a sequence having at least 90% sequence identity with the amino acids 402-533 of SEQ ID NO: 13;
the at least one inactivated chromosomal gene is inactivated by disruption;
and the host cell has reduced protease activity on the heterologous recombinant protein of interest in the host cell compared to protease activity on the heterologous recombinant protein of interest in the host cell without said inactivation, and wherein the spent culture broth has a reduced amount of endogenous proteases compared to a corresponding spent culture broth produced in a host cell with an intact nucleic acid sequence encoding a polypeptide comprising a sequence having at least 90% sequence identity with the amino acids 402-533 of SEQ ID NO: 13;
and wherein the heterologous recombinant protein of interest has an increased stability in the spent culture broth compared to a protein preparation produced in the same host cell without said inactivation.