Codon Optimization & Verification

The demand for recombinant proteins has increased significantly during the past decades, this has in turn increased the need for producing sufficient amounts of high-quality protein samples for various applications. ProteinCT codon optimization and verification services are designed to facilitate the identification of optimal genetic combinations for enhancing recombinant protein expression.

With the availability of vast amount of genomics data, along with increased knowledge of protein expression, function and structure relationships, gene expression levels are correlated to many factors, such as codon usage, promoter sequences and regulatory elements. One of the most important among these factors is the adaptation of the codon usage of transcribed genes to the typical codon usage of the host. Especially for prokaryotic systems, prominent codon usage bias is found in highly expressed genes under translational selection. In eukaryotes especially mammalian systems, other factors, such as transcriptional silencing, mRNA destabilization, alternative splicing, premature polyadenylation, or inefficient translation, often compromise protein expression. Engineering the expression host (e.g. providing rare tRNA pools) or using improved expression cassettes with strong or tissue-specific promoters may result in enhanced protein expression in various systems, however, the key to protein expression in many hosts still remains gene-specific in most cases, therefore, direct modification of the coding sequence, i.e., codon optimization, remains the key step for the success in protein expression enhancement.

Many companies offer codon optimization/gene sequence optimization and synthesis of the expression constructs. ProteinCT Biotechnologies has taken this one step further - our codon optimization and verification services, taking consideration of published factors that may affect protein expression combined with years of empirical observations by our experienced staff, are tailored to each expression system. We will construct expression vectors, perform EXPERIMENTAL VALIDATION for you and delivery a comprehensive report.

Applications:

Express a novel protein target
Increase production level of a poorly expressed protein
Improve antibody production for downstream process development
Enhance protein expression level to reduce production cost

Key Features:

Significant improvement in expression: Combinatorial approach for success
Comprehensive coverage: Optimization for bacteria, yeast, mammalian and plant system
Guaranteed result: Full report delivered for each combination tested

Parameters considered but not limited to the following:

• Codon choice	• GC-content	• UpA and CpG-dinucleotide contents
• Cryptic splice-sites	• Direct repeats	• Intragenic poly(A)-sites
• Destabilizing RNA elements	• Internal ribosomal entry sites	• RNA secondary structure

Service Specifications:

Customer Provides	Service Specifications	Deliverables
• Target sequence (or accession number) to be optimized for • Expression system to be optimized for: E. coli, Pichia pastoris, mammalian cells (CHO/HEK293), insect cells (sf9), green algae • Desired number of constructs/sequences to be evaluated	1. Construct optimization*: Sequence Optimization; Gene Synthesis 2. Subcloning to expression vector(s) 3. Experimental verification: Protein expression screening; SDS PAGE analysis; Test affinity purification when applicable	• Full report including optimization of DNA sequences, protein expression screening analysis and our recommended constructs • >2µg DNA of expression optimized construct with sequence file • Professional consultation on data analysis • (Optional) Downstream protein expression/purification, scale up production, stable cell line development services are also available upon request

*Charges for construct optimization includes optimization with our proprietary software and gene synthesis *Subcloning step is free and included in the service package *Experimental verification service charge is expression system dependent

Case Studies:

Selection Guide:

Quote today for an estimate on service cost and turnaround time

Expression System	Service Types*	Turnaround Time
Bacteria - E. coli	• Single sequence evaluation • Four sequence combinatorial evaluation • Eight sequence combinatorial evaluation Verification charge starting from $300	5-8 weeks
Yeast - Pichia pastoris	• Single sequence evaluation • Four sequence combinatorial evaluation • Eight sequence combinatorial evaluation Verification charge starting from $500	5-8 weeks
Insect - Sf9 cells	• Single sequence evaluation • Four sequence combinatorial evaluation • Eight sequence combinatorial evaluation Verification charge starting from $1000	6-9 weeks
Mammalian cells - CHO/HEK293	• Single sequence evaluation • Four sequence combinatorial evaluation • Eight sequence combinatorial evaluation Verification charge starting from $1000	6-9 weeks
Green Algae	• Single sequence evaluation • Four sequence combinatorial evaluation • Eight sequence combinatorial evaluation Verification charge starting from $1500	10-15 weeks