High level stable expression of recombinant HIV gp120 in glutamine synthetase gene deficient HEK293T cells

https://doi.org/10.1016/j.pep.2021.105837Get rights and content

Highlights

  • We developed an amplifiable stable human cell line for recombinant protein expression.

  • The glutamine synthetase-based expression system allows recombinant plasmid amplification by increasing MSX concentration.

  • Through signal peptide replacement and codon optimization, we have greatly improved the expression of HIV gp120.

  • The expression system retains human glycans.

  • The expression system is generally applicable to other glycoproteins.

Abstract

Due to the important pathological roles of the HIV-1 gp120, the protein has been intensively used in the research of HIV. However, recombinant gp120 preparation has proven to be difficult because of extremely low expression levels. In order to facilitate gp120 expression, previous methods predominantly involved the replacement of native signal peptide with a heterologous one, resulting in very limited improvement. Currently, preparation of recombinant gp120 with native glycans relies solely on transient expression systems, which are not amendable for large scale production. In this work, we employed a different approach for gp120 expression. Besides replacing the native gp120 signal peptide with that of rat serum albumin and optimizing its codon usage, we generated a stable gp120-expressing cell line in a glutamine synthetase knockout HEK293T cell line that we established for the purpose of amplification of recombinant gene expressions. The combined usage of these techniques dramatically increased gp120 expression levels and yielded a functional product with human cell derived glycan. This method may be applicable to large scale preparation of other viral envelope proteins, such as that of the emerging SARS-CoV-2, or other glycoproteins which require the presence of authentic human glycans.

Introduction

HIV-1 envelope glycoprotein forms a heterotrimeric gp120 and gp41 complex. Gp120 binds to host CD62L and CD4 receptors to initiate HIV entry through chemokine receptors [1,2]. It consists of five conserved (C1–C5) and five variable (V1–V5) regions [3], with the conserved regions forming the core of gp120 and the variable regions forming disulfide-bonded surface loops [4]. In addition to gp120's function in viral entry into the host cell, it is becoming increasingly clear that gp120 also contributes greatly to the pathogenesis of HIV infection. For example, gp120 activates complement, induces polyclonal B cells activation, binds into immune complexes, and influences T cell function [5,6]. The role of gp120 in HIV infection makes it a prime focus for vaccine development and the protein has been intensively used in the research of HIV [7]. However, recombinant gp120 preparation is challenging due to its low expressions [8]. While the native gp120 signal peptide (SP), which directs the nascent polypeptide to endoplasmic reticulum, may be responsible for the low expression of gp120(9), replacing it resulted in very limited improvement for the protein's production [[9], [10], [11], [12]].

Unlike many viral surface proteins, gp120 is a heavily glycosylated protein with more than 20 N-linked glycans of both high-mannose and complex branched-type, contributing to ~50% of the apparent molecular mass of the protein [[13], [14], [15]]. Carbohydrates on gp120 not only are essential for correct folding and intracellular transport of the protein [9,16,17], but also contribute to interactions with host cellular receptor molecules [2,[18], [19], [20]]. In addition, many neutralizing antibodies recognize gp120 glycans. The importance of the viral glycan in HIV pathogenesis and vaccine development obligated a human cell-base expression system for the production of recombinant gp120 in order to preserve the glycan function. Previously, both insect cells and CHO cells have been used to express gp120. Insect cells are known to have different protein glycosylation capabilities [21]. CHO cells, while producing mammalian glycans, differ in several aspects in glycan structures from human cells [22,23].

Currently, majority of recombinant gp120 for vaccine development is produced in human HEK293 cells using transient transfections. Despite improvements in the HEK293-based expressions, transient expression has distinct short comings, such as its high cost, low expression yield and difficultly in scaling up for large productions. These shortcomings, in general, can be mitigated by using stable transfection-based expression systems. One of the widely used stable expression system is based on glutamine synthetase (GS) expression under selection of methionine sulfoximine (MSX) [24]. However, currently available GS expression systems are non-human cell lines, such as CHO and NS0 [25,26].

To generate a human cell line-based GS expression system, we carried out CRISPR/Cas9 knockout of GS gene in HEK293T cells, referred to as 293T-GS- cells. A stable gp120-expressing cell line was then generated by transfecting a codon optimized gp120 with non-native signal peptide into 293T-GS- cells. This combination dramatically increased recombinant gp120 expression levels and produced a functional product with glycan features different from those of gp120 derived from CHO cells.

Section snippets

Reagents

Trizma@ hydrochloride, Trizma@ base, ammonium bicarbonate, urea, tris (2-carboxyethyl) phosphine hydrochloride (TCEP), iodoacetamide (IAM), and glacial acetic acid were purchased from Sigma-Aldrich (St. Louis, MO). Other reagents used in this study included optima LC/MS grade acetonitrile, water, formic acid (Thermo Fisher Scientific, Atlanta, GA), sequencing grade trypsin, chymotrypsin (Promega, Madison, WI), and glycerol-free peptidyl-N-glycosidase F (PNGase F) (New England BioLabs, Ipswich,

Establishment of a GS-deficient HEK293T cell line

HEK293T cells were co-transfected with pGLu-sgRNA and pSpCas9(BB)-2A-GFP, which code gRNA targeting human GS gene and Cas9 respectively. After selection with puromycin and growth testing in glutamine-free medium, clones that became completely dependent on the addition of glutamine were isolated for further evaluation in glutamine-free GMEM medium with or without glutamine. Ten thousand HEK293T or GS deficient clone cells were cultured in 6-well plates with 5 mL of glutamine-free GMEM

Discussion

Recombinant gp120 was difficult to express in part due to its signal peptide composition [8]. The presence of positively charged amino acids in HIV-1 env signal sequence may affect the rate of SP cleavage, resulting in less efficient protein folding, intracellular transport, and secretion [9,12]. In addition, the SP may associate with calnexin to deter glycoprotein folding [11]. These findings support the replacement of the native gpl20 signal sequence with a heterologous one to promote gp120

CRediT authorship contribution statement

Zhongcheng Zou: Conceptualization, Methodology, Formal analysis, Investigation. Ruipeng Wang: Investigation. Eden P. Go: Investigation. Heather Desaire: Resources, Supervision, Funding acquisition. Peter D. Sun: Conceptualization, Supervision, Funding acquisition, Writing - review & editing.

Acknowledgements

This research was supported by the NIH Intramural Research Program and NIH grant R01AI125093. We thank Hursch Patel for his assistance in the manuscript preparation.

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