B. are expressed in CHO cells, they are proteolyzed and lack important glycan-dependent epitopes present on virions. Previously, we identified C1s, a CHIR-98014 serine protease in the complement pathway, as the endogenous CHO protease responsible for the cleavage of clade B laboratory isolates of -recombinant gp120s (rgp120s) expressed in stable CHO-S cell lines. In this paper, we describe the development of two novel CHOK1 cell lines with the C1s gene inactivated by gene editing, that are suitable for the production of any protein susceptible to C1s proteolysis. One cell line, C1s-/- CHOK1 2.E7, contains a deletion in the C1s gene. The other cell line, C1s-/- MGAT1- CHOK1 1.A1, contains a deletion in both the C1s gene and the MGAT1 gene, which limits glycosylation to mannose-5 or earlier intermediates in the N-linked glycosylation pathway. In addition, we compare CHIR-98014 the substrate specificity of C1s with thrombin on the cleavage of both rgp120 and human Factor VIII, two recombinant proteins known to undergo unintended proteolysis (clipping) when expressed in CHO cells. Finally, we demonstrate the utility and practicality CHIR-98014 of the C1s-/- MGAT1- CHOK1 1.A1 cell line for the expression of clinical isolates of clade B Envs from rare individuals that possess broadly neutralizing antibodies and are able to control virus replication without anti-retroviral drugs (elite neutralizer/controller phenotypes). The Envs represent unique HIV vaccine immunogens suitable for further immunogenicity and efficacy studies. Introduction The majority of recombinant glycoprotein therapeutics are manufactured in CHO (Chinese Hamster Ovary) cells due to their high productivity (1C10 grams per liter), genetic stability, and ability to be grown in large-scale suspension culture [1C3]. However, many recombinant proteins including monoclonal antibodies, CHIR-98014 antibody fusion proteins, and IFN- are partially degraded or clipped by endogenous CHO cell proteases during the cell culture or recovery process [4C9]. This is also the case for glycoprotein 120 (gp120), the monomeric subunit of the HIV-1 envelope protein (Env), used in many of the HIV vaccines tested to date in human vaccine efficacy trials [10C13]. The HIV Env protein mediates virion binding to CD4, the T-cell surface receptor, and to the CXCR4 or CCR5 chemokine receptors [14C16]. Env proteins have been included in most HIV vaccines since they are the major target for virus neutralizing antibodies [17C19]. HIV Rabbit Polyclonal to UTP14A isolates are classified into different genetic clades based on impartial sequence analysis [20,21]. These include clades C and CFRF01_AE viruses, prevalent in Africa and Asia respectively, and clade B viruses in North America, Europe, the Caribbean and Australia. Because they lack the clade B consensus sequence Gly-Pro-Gly-Arg-Ala-Phe (GPGR/AF) at the crown of the V3 domain, most clade C and CRF01_AE Envs can be produced in CHO cells without proteolysis. In contrast, the V3 domain of most clade B Envs has been shown to be highly sensitive to proteolysis by exogenous thrombin or an unidentified CHO cell protease [22C25]. Env proteins proteolyzed in this manner are difficult to manufacture and purify in quantities required for immunization of populations at high risk for infection [24,26]. Recently, we reported that the major CHO cell protease responsible for cleavage of clade B gp120s was the complement component 1 protease, C1s . C1s is a serine protease that recognizes the sequence Gly-Pro-Gly-Arg, located in the V3 loop of gp120. This sequence is present in 71% of clade B HIV strains  and is also present in the Env protein.
Posted by By stemcellresearchformichigan January 21, 2022
However, most of these studies use the MDA-MB-231 cell line mainly because the TNBC tumor model and, only occasionally compare it to another cell line