Rapid preparation of proteins for functional and structural analysis is a major challenge both in academia and industry. The number potential targets continuously increases and many are difficult to express proteins which, when produced in bacteria, result in insoluble and/or misfolded recombinant proteins, protein aggregates, or unusable low protein yield. We focus here on the baculovirus expression vector system which is now commonly used for heterologous production of human targets. This chapter describes simple and cost-effective protocols that enable iterative cycles of construct design, expression screening and optimization of protein production. We detail time- and cost-effective methods for generation of baculoviruses by www.joplink.net/baculovirus-recombinant/ homologous recombination and titer evaluation. Handling of insect cell cultures and preparation of bacmid for cotransfection are also presented.

BacMam system utilizes baculovirus to deliver exogenous genes into mammalian cells and is extensively used for recombinant production of eukaryotic proteins. Here, we described the development of a BacMam vector (pBMCL1), which allows convenient tracing of virus production, provides higher infection efficiency towards mammalian cells, minimizes unwanted transcription of toxic genes in insect cells, and provides the capability for co-expression of multiple proteins via a single virus. We demonstrate the successful application of the pBMCL1 vector for the expression of homo-tetrameric human TRPC3 channel and hetero-octameric K_ATP channel.

The baculovirus display system (BDS), an excellent eukaryotic surface display technology that offers the advantages of safety, efficiency, and economy, is widely used in biomedicine. A previous study using rBacmid-Δgp64-ires-gp64 expressed in low copy numbers of the gp64 gene achieved high-efficiency expression and co-display of three fluorescent proteins (GFP, YFP, and mCherry). However, low expression of GP64 in recombinant baculoviruses also reduces the efficiency of recombinant baculovirus transduction into mammalian cells.

In addition, the baculovirus promoter has no expression activity in mammalian cells and thus cannot meet the application requirements of baculoviral vectors for the BDS. Based on previous research, this study first determined the expression activity of promoters in insect Spodoptera frugiperda 9 cells and mammalian cells and successfully screened the very early promoter pie1 to mediate the co-expression of multiple genes. Second, utilizing the envelope display effect of the INVASIN and VSVG proteins, the efficiency of transduction of recombinant baculovirus particles into non-host cells was significantly improved. Finally, based on the above improvement, a recombinant baculovirus vector displaying four antigen proteins with high efficiency was constructed.

Compared with traditional BDSs, the rBacmid-Δgp64 system exhibited increased display efficiency of the target protein by approximately 3-fold and induced an approximately 4-fold increase in the titer of serum antibodies to target antigens in Bal B/c mice. This study systematically explored the application of a new multi-gene co-display technology applicable to multi-vaccine research, and the results provide a foundation for the development of novel BDS technologies.

The species-selective interaction between sperm and egg at the beginning of mammalian fertilisation is partly mediated by a transparent envelope called the zona pellucida (ZP). The ZP is composed of three or four glycoproteins (ZP1-ZP4). The functions of the three proteins present in mice (ZP1-ZP3) have been extensively studied. However, the biological role of ZP4, which was found in all other mammals studied so far, has remained largely unknown. Previously, by developing a solid support assay system, we showed that ZP4 exhibits sperm-binding activity in bovines and the N-terminal domain of bovine ZP4 (bZP4 ZP-N1 domain) is a sperm-binding region.

Here, we show that bovine sperm bind to the bZP4 ZP-N1 domain in a species-selective manner and that N-glycosylation is not required for sperm-binding activity. Moreover, we identified three sites involved in sperm binding (site I: from Gln-41 to Pro-46, site II: from Leu-65 to Ser-68 and site III: from Thr-108 to Ile-123) in the bZP4 ZP-N1 domain using chimeric bovine/porcine and bovine/human ZP4 recombinant proteins. These results provide in vitro experimental evidence for the role of the bZP4 ZP-N1 domain in mediating sperm binding to the ZP.

Protein semisynthesis has been used for the chemoselective linking of synthetic peptides and recombinant protein fragments to generate complete native proteins in good yield. The ability to site-selectively incorporate multiple post-translational chemical modifications (PTMs) into proteins via this approach shows great potential for enhancing understanding of the molecular basis of protein function and regulation.

Protein semisynthesis, however, often requires high expression efficiency of the recombinant protein fragments (i.e., high expression yield and ability to preserve protein biological functions), which can be hard to achieve for some human enzymes when using bacterial expression systems. Here, we describe how to use a baculovirus/insect cell expression system and a protein semisynthesis strategy known as expressed protein ligation (EPL) to produce workable levels of proteins of interest containing site-specific chemical modifications. The protocol provides detailed guidance for generating protein C-terminal thioesters for use with the EPL reaction, performing the EPL reaction, and purifying the protein ligation product.

We exemplify the protocols by generating protein kinase Akt1 with site-specific phosphorylations installed into its C-terminal tail, for kinetic kinase assays. We hope these methods will help increase the use of protein semisynthesis for elucidating the post-translational regulation of human enzymes involved in cell signaling. © 2022 Wiley Periodicals LLC Basic Protocol 1: Generation of the N-terminal protein of interest (POI) fragment containing a C-terminal thioester moiety Basic Protocol 2: Expressed protein ligation (EPL) of the protein thioester with a synthetic peptide and purification of the protein ligation product Basic Protocol 3: Semisynthesis and biochemical analysis of site-specifically phosphorylated Akt1.